INDEX. III. Preparative Packing Materials I. HPLC Columns...4. IV. Related Products II. SFC Columns...55

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3 IDEX CSMSIL / CSMGEL Packing Material List... 2 Column Selection Guide... 3 I. HPLC Columns Core-Shell Columns... 4 (1) CSMCRE Series... 4 CSMCRE 2.6C CSMCRE 2.6Cholester... 8 CSMCRE 2.6PBr (2) Instrument Settings and Compatibility HPLC Columns (1) Reversed Phase Columns C18 (DS) Series CSMSIL C18-MS-II CSMSIL C18-AR-II CSMSIL C18-PAQ CSMSIL C18-EB Reversed Phase Specialty Columns CSMSIL Cholester CSMSIL PBr CSMSIL AP CSMSIL PYE CSMSIL PE ther Reversed Phase Columns CSMSIL PFP CSMSIL C -MS CSMSIL C22-AR-II, C8-MS, C4-MS, TMS-MS, PE-MS (2) ormal Phase Columns CSMSIL SL-II (3) Hydrophilic Interaction Columns CSMSIL HILIC (4) Mono- and ligosaccharide Analysis Columns CSMSIL Sugar-D CSMSIL H2-MS (5) Protein Separation Columns Reversed Phase Columns CSMSIL Protein-R CSMSIL C18-AR-300, C8-AR-300, C4-AR-300, Ph-AR Gel Filtration Columns (Aqueous) CSMSIL Diol-120-II, Diol-300-II, Diol-1000-II Ion Exchange Columns CSMGEL IEX Series Hydrophobic Interaction Columns CSMSIL HIC (6) Columns for Fullerene Separation CSMSIL Buckyprep CSMSIL Buckyprep-D CSMSIL Buckyprep-M CSMSIL PBB CSMSIL PE CSMSIL PYE (7) Columns for Soluble Carbon anotube Separation CSMSIL C T-300, C T-1000, C T II. SFC Columns...55 CSMSIL HP (3-Hydroxyphenyl) CSMSIL PY (Pyridinyl) CSMSIL Quinoline CSMSIL Cholester CSMSIL PBr III. Preparative Packing Materials...60 ormal and Reversed Phase Packing Materials CSMSIL C18-P CSMSIL C18-PREP Silica Gel (Spherical, eutral) Silica Gel (for Column Chromatography) IV. Related Products Reagents for Mobile Phase Preparation Phosphate Buffer Solution (ph 2.5) (5X) Stock Solutions for HPLC Premixed Eluents for HPLC Additives Ion-pair Reagents Products for Sample Preparation Cosmonice Filter Cosmospin Filter Labeling Reagents Column Care Products Cleaning Solution Kit for Reversed Phase HPLC Columns Storage Solution for Reversed Phase HPLC Columns CSMSIL HPLC Accessories

4 CSMSIL / CSMGEL Packing Material List Core-Shell Columns Sample rganic compounds (low M.W.) Separation Mode Reversed phase Packing Material Bonded Phase Bonding Type C18 ctadecyl group Polymeric Cholester PBr Cholesteryl group Pentabromobenzyl group Average Particle Si e ( m) Average Pore Size ( ) Carbon Content (%) Special Features and Applications USP Category Page 7 Multi-purpose C18 column L1 5, 6 Usable under the same condition as C18. Unique rigid cholesteryl L101 5, 8 - structure improves separation. Separate hydrophilic compounds under reversed-phase conditions. - 5,10 HPLC Columns 2 Sample rganic compounds (low M.W.) Separation Mode Reversed phase ormal phase Mono- and Hydrophilic ligosaccharides interaction Proteins Packing Material C18-MS-II Bonded Phase Bonding Type Average Particle Size ( m) Average Pore Size ( ) Carbon Content (%) , 5, C18-AR-II 3, 5,15 17 ctadecyl group Polymeric 120 C18-PAQ 5,15 11 C18-EB Cholester PBr Cholesteryl group Pentabromobenzyl group Monomeric Monomeric Monomeric AP aphthylethyl group PYE Pyrenylethyl group Monomeric PE itrophenylethyl group 9 PFP Pentafluorophenyl group 10 Special Features and Applications Multi-purpose C18 column Features strong acid resistance, good for acidic compounds and peptides. Good for hydrophilic compounds, and stable performance under 100% aqueous conditions. USP Category L1 Page 14, 15 14, 17 14, Good for basic compounds 14, Usable under the same conditions as C18. Unique rigid cholesteryl L101 22, structure improves separation. Separate hydrophilic compounds , 25 under reversed-phase conditions Stronger - interaction than 22, 26 phenyl column The most powerful - interaction 22, 28 Separation utilizing dipole-dipole interaction. Separation utilizing weak dipoledipole interaction. 22, 29 L43 30, 31 Enables separation of different C -MS Cyanopropyl group 7 hydrophobic samples without using L10 30, gradients. C22-AR-II Docosyl group Polymeric 19 - C8-MS ctyl group 10 Alkyl chain columns, excluding C18 L7 C4-MS Butyl group Monomeric 7 column L26 30, 33 TMS-MS Trimethyl group 5 L13 PE-MS Phenylethyl group 10 - interaction L11 SL-II , 5, Suitable for preparative separation. L3 34 Hydrophilic interaction HILIC Triazole Reversed phase Gel permeation Ionexchange Hydrophobic interaction - Retains highly polar compounds that would not be retained in a C18 olumn. A novel stationary phase for monoand oligosaccharides. L Sugar-D Secondary/Tertiary - amine , 38 H2-MS Aminopropyl group Polymeric Primary amino bonded column L8 37, 39 Protein-R Wide-pore column with the - ctadecyl group advantages of both C18 and C4 L1 40 C18-AR L1 Polymeric C8-AR-300 ctyl group L7 Wide pore type C4-AR-300 Butyl group 6 L26 41 Ph-AR-300 Phenyl group 7 L11 Diol-120-II 120 Diol-300-II Diol group Diol-1000-II 1000 IEX Type Q 1000 Trimethylaminopropyl IEX Type Q- type - - Silica-based gel filtration column Sample MW (Protein) 5, ,000 Da Silica-based gel filtration column Sample MW (Protein) 10, ,000 Da Silica-based gel filtration column Sample MW (Water Soluble Polymer) 50, ,000 Da Anion-exchange type (purification) Anion-exchange type (ultra-fast analysis, precise analysis IEX Type S 1000 Cation-exchange type (purification) IEX Type S- Sulfopropyl type Cation-exchange type (ultra-fast analysis, precise analysis IEX Type M Amphoteric ion-exchange type 1000 Trimethylaminopropyl (purification) type /Sulfopropyl type Amphoteric ion-exchange type IEX Type M- - (precise analysis) HIC Little loss in enzyme activity and the tertiary structure of proteins L

5 Sample Fullerenes -- Carbon nanotubes Separation Mode Gel permeation Packing Material Bonded Phase Bonding Type Average Particle Size ( m) Average Pore Size ( ) Carbon Content (%) Special Features and Applications USP Category Page SFC Columns Sample - SFC Separation Mode Packing Material HP PY Bonded Phase 3-Hydroxyphenyl group Pyridinyl group Quinoline Quinoline group Cholester PBr Cholesteryl group Pentabromobenzyl group Bonding Type Average Particle Size ( m) Average Pore Size ( ) Carbon Content (%) Special Features and Applications USP Category Page Buckyprep Pyrenylpropyl group 17 Standard column for fullerene separation. 48, 49 Buckyprep-D itro-carbazoyl group - Good for derivatized fullerenes 48, 50 Buckyprep-M Phenothiazinyl group Monomeric Good for preparative separation of 13 Good for metallofullerenes 48, Pentabromobenzyl PBB 8 48, 52 group C60 or C70. PE itrophenylethyl group 9 Separation of derivatized fullerenes 48, 53 PYE Pyrenylethyl group 18 Separation of fullerenes 48, 52 C T Hydrophilic group Separation of soluble carbon C T (neutral) nanotubes. C T Good for hydrophilic compounds. Stronger retention for basic 55, 56 Polymeric 3, compounds than PY - Similar separation properties as - 2-Ethylpyridine, with stronger 55, 57 retention. 2.5, Alternate selectivity to HP and PY. 55, Usable under the same conditions Monomeric structure improves separation. as C18. Unique rigid cholesteryl L101 55, Separate hydrophilic compounds under reversed-phase conditions. 55, 59 Column Selection Guide Selection by Stationary Phase Type ot enough separation Usable under the same conditions as C18 Increased stereoselectivity Stronger π-π interaction than phenyl column. The most poweful π-π interaction First-choice column C18 (DS) Pentafluorophenyl Pentabromobenzyl Poor peak shape o retention Separate hydrophilic compounds under reversed-phase conditions o retention on reversed-phase (Hydrophilic compounds) Good for basic compounds Specialty columns Selection by Particle Size First-choice column C18 (DS) 5 μm Shorten analysis time Increase resolution Lower back pressure than CSMCRE 2.6C18 Can be used with any conventional LC system Improved endcapping for high resolution Can be used with any conventional LC system Better resolution than 2.5C18-MS-II 3

6 I. HPLC Columns II. SFC Columns I. HPLC Columns 1. Core-Shell Columns (1) CSMCRE Series About Core-Shell Particles Core-shell particles consist of a nonporous core inside a porous shell. By using these core-shell particles, one can achieve sharper peaks compared to fully porous silica gel particles of the same diameter. Uniform Particle Size Distribution Compared to 1.7 μm Particles Compared to fully porous particles, core-shell particles have a more uniform particle diameter; therefore, core-shell particles can be packed in the column more uniformly to minimize sample diffusion. 2.6 μm Schematic diagram of a silica particle 0.5 μm 1.6 μm III. Preparative Packing Materials 10 μ CSMCRE 2.6C18 (200x) 10 μ Fully porous 1.7 μm particles (200x) Mass Transfer Equivalent to Fully Porous sub-2 m Particles Mass transfer refers to the time it takes for a sample molecule to enter and leave a particle. In general, lower mass transfer time corresponds to less diffusion and sharper peaks. Even though CSMCRE 2.6C18 has a larger particle diameter than fully porous sub-2 μm particles, the mass transfer characteristics are similar. IV. Related Products CSMCRE 2.6C18 Fully porous sub-2 μm particle Reduced Back Pressure and Faster Analyses CSMCRE 2.6C18 delivers performance equivalent to sub-2 μm particles at faster flow rate and analysis time while maintaining a lower back pressure. CSMCRE can also be used in longer column size to gain additional resolution. Column pressure (MPa) Pressure vs. flow rate (70% AC / 30% water) Pressure Plot CSMCRE 2.6C18 Fully porous, 1.7 μm, C18 Theoretical plate height (μm) Theoretical plate height vs. flow rate van Deemter Plot Flow rate (ml / min) Flow rate (ml / min) Column size: 2.1 mm I.D. x 50 mm Temperature: 40 o C Mobile phase: Acetonitrile / Water = 70 / 30 Amylbenzene 4

7 Same performance and lower back pressure compared to sub-2 μm particles Reduced Back Pressure CSMCRE 2.6C18 maintains the same performance as sub-2 μm particles with half the back pressure. Speci cations Fully porous C18, 1.7 μm Company A C18 Flow rate 0.4 ml/min Pressure 39.3 MPa (Peak 6) plates Column: Fully porous, 1.7 μm: Company A, 1.7 μm, C18 Core-Shell, 2.6 μm: CSMCRE 2.6C18 Column size: 2.1 mm I.D.-100 mm Mobile phase: Acetonitrile / Water = 60 / 40 Temperature: 40 C Detection: UV254nm 1; Benzene (2.0 mg/ml) 2; Toluene (2.0 mg/ml) 3; Ethylbenzene (2.0 mg/ml) 4; Propylbenzene (2.0 mg/ml) 5; Butylbenzene (2.0 mg/ml) 6; Amylbenzene (2.0 mg/ml) Inj.Vol.: 0.5 μl Same separation with half the pressure Half the analysis time while minimizing loss of separation 3.6 min Core-Shell C18, 2.6 μm CSMCRE 2.6C18 Flow rate 0.4 ml/min Pressure 17.7 MPa (Peak 6) plates 6.9 min Pressure 36.0 MPa (Peak 6) plates Packing Material 2.6C18 2.6Cholester 2.6PBr Silica Gel Core-shell type silica gel Average Particle Size 2.6 m Average Core Diameter 1.6 m Average Pore Size approx. 90 Speci c Surface Area approx. 150 m 2 /g H3C Core-Shell C18, 2.6 μm CSMCRE 2.6C18 Flow rate 0.8 ml/min Br Br Br I. HPLC Columns II. SFC Columns III. Preparative Packing Materials IV. Related Products Bonded Phase Structure H3C Br Br Si H Bonded Phase ctadecyl group Cholesteryl group Pentabromobenzyl Group Main interaction Hydrophobic interaction Hydrophobic interaction Molecular shape selectivity End-Capping Treatment ear-perfect treatment Usable ph Range Maximum Pressure 60 MPa H3C Si CH3 H3C Si CH3 Hydrophobic interaction Dispersion force 5

8 CSMCRE 2.6C18 IV. Related Products III. Preparative Packing Materials II. SFC Columns I. HPLC Columns Ultra-high performance LC results with conventional HPLC equipment Same number of theoretical plates as sub-2 μm columns with half the back pressure Increased loading capacity Excellent ph stability (1.5-10) Excellent ph Stability Under accelerated ph 10.4, 40 C stability test, CSMCRE C18 shows superior stability compared with other core-shell C18 phases Column size: 2.1 mm x 100 mm Mobile phase: 0.35% Ammonium hydroxide/ acetonitrile = 90/10 (ph 10.4) Caffeine (0.05 mg/ml) Injection volume: 1 μl Flow rate: 0.4 ml/min. Temperature: 40 C Sharp Peaks with Many Types of Compounds CSMCRE 2.6C18 features a special end capping treatment that effectively shields residual silanol groups, yielding sharp peaks for basic compounds and metal coordination complexes. Basic Compounds Amitriptyline High ph Stability Test CSMCRE C μm Injection 1 Injection 300 Injection 1,000 Column size: 2.1 mm I.D.- 50 mm Mobile phase: Acetonitrile / 20mmol/l Phosphoric Buffer(pH7.0) = 60 / 40 Temperature: 40oC Flow rate: 0.4 ml/min Detection: UV254nm 1; Amitriptyline (0.2 mg/ml) 2; Amylbenzene I.S. (2.0 mg/ml) Inj.Vol.: 0.5 μl Amitriptyline Haloperidol Column size: 2.1 mm I.D mm Mobile phase: A; Acetonitrile/ 20mmol/l Phosphate buffer(ph7.0) = 10/90 B; Acetonitrile/ 20mmol/l Phosphate buffer(ph7.0) = 50/50 B conc %(0 3min), 100%(3-5min) Flow rate: 0.4 ml/min Temperature: 40oC Detection: UV220nm Inj. Vol: Cl H Haloperidol 1; 4-(4-Chlorophenyl)-4-hydroxypiperidine (0.1 mg/ml) 2; Haloperidol (0.1 mg/ml) 0.5 μl High ph Stability Test Company P C μm Injection 1 Injection 300 Injection 1,000 F Procainamide Column size: 2.1 mm I.D mm Mobile phase: Acetonitrile/ 20mmol/l Phosphate buffer(ph7.0) = 10/90 Flow rate: 0.4 ml/min Temperature: 40oC Detection: UV270nm 1; Procainamide (0.05 mg/ml) 2; -Acetylprocainamide (0.05 mg/ml) Inj. Vol: 0.5 μl Caffeine Column size: 2.1 mm I.D mm Mobile phase: Acetonitrile/ H2 = 10/90 Flow rate: 0.4 ml/min Temperature: 40oC Detection: UV275nm 1; Theobromine (0.05 mg/ml) 2; Theophylline (0.05 mg/ml) 3; Caffeine (0.05 mg/ml) Inj.Vol.: 1.0 μl H 2 H H 3 C Procainamide Caffeine 6

9 Metal Coordination Complexes xine-copper Column size: 2.1 mm I.D.-50 mm Mobile phase: Acetonitrile / 20mmol/l Phosphoric Acid = 10 / 90 Temperature: 40 C Flow rate: 0.4 ml/min Detection: UV254nm 1; xine-copper (0.003 mg/ml) 2; Caffeine (I.S.) (0.3 mg/ml) Inj.Vol.: 0.5 μl Cu xine-copper Low Bleed-Suitable for LC-MS CSMCRE 2.6C18 has low column bleed and consequently low MS noise level. Column size: Mobile phase: rdering Information 2.1 mm I.D.-50 mm A: 0.1% Formic Acid B: Acetonitrile B conc % (0-5 min), 100% (5-10 min), 100 5% (10-11 min), 5% (11-17 min) 0.2 ml/min Flow rate: Temperature: 40 C Detection: MS (TIC (+), MW ) Analytical Columns (Particle Size: 2.6 m) CSMCRE 2.6C18 Packed Columns 2.1 x x x x x CSMCRE's connector is the same type as Waters UPLC columns. For UHPLC-compatible pre lters, refer to page 13. without Column 3.0 x x x x x Core-Shell C18, 2.6 μm CSMCRE 2.6C18 Core-Shell C18, 2.6 μm Company B C x x x x x x I. HPLC Columns II. SFC Columns III. Preparative Packing Materials IV. Related Products 7

10 CSMCRE 2.6Cholester I. HPLC Columns Cholesterol-bonded reversed-phase core-shell column Usable under the same conditions as C18 columns Better selectivity for cis-trans isomers, polyphenols, and natural products Comparison with C18 CSMCRE 2.6Cholester offers improved separation for cis-trans isomers than C18 under typical reversed-phase mobile phase. CSMCRE Application Data Competitor A Competitor B CSMCRE CSMCRE II. SFC Columns Column: Column size: 2.1mmI.D.-100mm Mobile phase: Methanol Flow rate: 0.4 ml/min Temperature: 40 C Detection: UV280nm 1; Vitamin K 2 (0.50mg/ml) 2; trans-vitamin K 1 (0.50mg/ml) 3; cis-vitamin K 1 Inj. Vol: 0.5μl (Fully porous, 1.7μm, C 18 ) (Core-Shell, 2.6μm, C 18 ) 2.6C Cholester H 3C trans-vitamin K 1 III. Preparative Packing Materials IV. Related Products Separation Properties CSMCRE 2.6Cholester has about the same hydrophobicity as C18. It is not necessary to change the analytical conditions when replacing C18 Columns with CSMCRE 2.6Cholester. CSMCRE Application Data Column: Column size: 2.1mmI.D.-100mm Mobile phase: Acetonitrile/ H 2 = 50/50 Flow rate: 0.4 ml/min Temperature: 40 C Detection: UV254nm Inj. Vol: 1; Uracil 2; Methyl Benzoate 3; Toluene 4; aphthalene 0.25μl Molecular Shape Selectivity Competitor A (Fully porous, 1.7μm, C 18 ) Competitor B (Core-Shell, 2.6μm, C 18 ) CSMCRE CSMCRE 2.6C Cholester CSMCRE 2.6Cholester has excellent shape slectivity due to its structural rigidity. CSMCRE 2.6Cholester retains planar triphenylene longer than non planar o-terphenyl. CSMCRE Application Data Column: Column size: 2.1mmI.D.-100mm Mobile phase: Acetonitrile/ H 2 = 70/30 Flow rate: 0.4 ml/min Temperature: 40 C Detection: UV254nm 1; o-terphenyl (0.25mg/ml) 2; Triphenylene (0.05mg/ml) Inj. Vol: 0.5μl Competitor A (Fully porous, 1.7μm, C 18 ) Competitor B (Core-Shell, 2.6μm, C 18 ) CSMCRE 2.6C 18 CSMCRE 2.6Cholester α = 1.13 α = 1.09 α = 1.16 α = 2.16 o-terphenyl Triphenylene 8

11 Applications Vitamin D metabolites CSMCRE Application Data Column: CSMCRE 2.6Cholester Column size: 2.1mmI.D.-150mm Mobile phase: A; 0.1% Formic acid-h 2 B; Methanol B conc % (0 11min), 95% (11 25min) Flow rate: 0.3 ml/min Temperature: 50 C Detection: UV265nm 1; 25-Hydroxyvitamin D 3 2; 3-epi-25-Hydroxyvitamin D 3 3; 25-Hydroxyvitamin D 2 4; 3-epi-25-Hydroxyvitamin D 2 5; Vitamin D 2 [Calciferol] 6: Vitamin D 3 Data courtesy of a customer rdering Information Analytical Columns (Particle Size: 2.6 m) CSMCRE 2.6Cholester Packed Column 2.1 x x x x x AP-1514 CSMCRE s connector is the same type as Waters UPLC columns. For UHPLC-compatible pre lters, refer to page 13. Catechins CSMCRE Application Data Column: CSMCRE 2.6Cholester Column size: 2.1mmI.D.-50mm Mobile phase: A; Acetonitrile/ 0.1% H 3 P 4 = 10/90 B; Acetonitrile/ 0.1% H 3 P 4 = 40/60 B conc % 3min Linear gradient Flow rate: 0.6 ml/min Temperature: 30 C Detection: UV280nm 1; (-)-Gallocatechin (-)-GC 2; Caffeine 3; (-)-Epigallocatechin (-)-EGC 4; (-)-Catechin (-)-C 5; (-)-Epicatechin (-)-EC 6; (-)-Epigallocatechin Gallate (-)-EGCg 7; (-)-Gallocatechin Gallate (-)-GCg 8; (-)-Epicatechin Gallate (-)-ECg 9; (-)-Catechin Gallate (-)-Cg Data courtesy of a customer 3.0 x x x x x x x x x x x I. HPLC Columns II. SFC Columns III. Preparative Packing Materials IV. Related Products 9

12 CSMCRE 2.6PBr I. HPLC Columns Separate hydrophilic compounds under reversed-phase conditions Retain hydrophilic compounds longer than C18 Greater sample loading capacity than HILIC High performance similar to sub-2 m particles with lower back pressure < Suitable Samples > Hydrophilic compounds ucleic acids and derivatives Surfactants Glycosides Peptides II. SFC Columns Separation of Hydrophilic Compounds (low retention on C18) CSMCRE 2.6PBr retains hydrophilic compounds stronger than C18 columns under the same reversed-phase conditions. ucleic Acid Metabolites Malonyl CoA, CoA, Acetyl CoA CSMCRE Application Data CSMCRE Application Data III. Preparative Packing Materials Column: CSMCRE ** Column size: 2.1mmI.D.-100mm Mobile phase: Methanol/ 100mmol/l Phosphate buffer(ph2.5) = 10/90 Flow rate: 0.4 ml/min Temperature: 40 C H Detection: UV240nm H 2 1; Allantoin (2.0mg/ml) 2; Guanine (0.05mg/ml) 3; Hypoxanthine (0.05mg/ml) H H 1; Allantoin 4; Uric Acid (0.1mg/ml) 5; Xanthine (0.2mg/ml) H 6; Adenosine (0.2mg/ml) H 2 7; Inosine (0.1mg/ml) H Inj. Vol: 1.0μl 2; Guanine CSMCRE 2.6C 18 CSMCRE 2.6PBr H H 3; Hypoxanthine H H H H 4; Uric Acid H H H 5; Xanthine H 2 H H H 6; Adenosine H H H H 7; Inosine Column: CSMCRE ** Column size: 2.1mmI.D.-100mm Mobile phase: Acetonitrile/ 20mmol/l Phosphate buffer(ph7.0) = 5/95 Flow rate: 0.4 ml/min Temperature: 40 C Detection: UV260nm Inj. Vol: CSMCRE 2.6C 18 1; Malonyl CoA (0.5mg/ml) 2; CoA (0.5mg/ml) 3; Acetyl CoA (0.5mg/ml) 1.0μl H HS CSMCRE 2.6PBr S H H H H H H H 3C H 3C H H P P - - 1; Malonyl CoA P 2; CoA - P - H 3C H 3C S P P H H - - H H - 3; Acetyl CoA - - H P - H P - H P - H 2 H 2 H 2 IV. Related Products k (Peak 1) = 0.00 k (Peak 1) = 0.51 Water-Soluble Vitamins CSMCRE Application Data Column: CSMCRE 2.6PBr Column size: 2.1mmI.D.-150mm Mobile phase: A; 20mmol/l Phosphate buffer(ph2.5) B; Methanol/ 20mmol/l Phosphate buffer(ph2.5) = 60/40 B conc. 0% (0-1min), 0 80% (1 5min), % (5 9min) Flow rate: 0.4 ml/min Temperature: 40 C Detection: UV220nm 1; Vitamin B 1 [Thiamine] (0.25mg/ml) 6; Vitamin B 5 [Pantothenic Acid] (2.0mg/ml) 2; Vitamin C [Ascorbic Acid] (1.0mg/ml) 7; Vitamin B 9 [Folic Acid] (0.2mg/ml) 3; Vitamin B 3 [icotinic Acid] (0.04mg/ml) 8; Vitamin B 7 [Biotin] (3.0mg/ml) 4; Vitamin B 3 [icotinamide] (0.06mg/ml) 9; Vitamin B 12 [Cyanocobalamin] (0.25mg/ml) 5; Vitamin B 6 [Pyridoxine] (0.25mg/ml) 10; Vitamin B 2 [Riboflavin] (0.2mg/ml) Inj. Vol: 1.25μl 10

13 Separation of Hydrophilic Compounds (compounds with similar hydrophobicity) CSMCRE 2.6PBr can separate compounds with similar hydrophobicity, utilizing several kinds of molecular interactions, including dispersion force generated by the bromine atoms. ATP ADP AMP Vitamin B3 (icotinic Acid, icotinamide) CSMCRE Application Data Column: CSMCRE ** Column size: 2.1mmI.D.-100mm Mobile phase: 20mmol/l Phosphate buffer(ph7.0) Flow rate: 0.4 ml/min Temperature: 40 C Detection: UV260nm 1; ATP (0.5mg/ml) 2; ADP (0.8mg/ml) 3; AMP (0.8mg/ml) Inj. Vol: 0.5μl Separation of Hydrophilic Compounds (Glycosides) - P - - P - P - - P - 1; ATP P - P - CSMCRE Application Data Column: CSMCRE 2.6PBr Column size: 2.1mmI.D.-150mm Mobile phase: 10mmol/l Phosphate buffer(ph7.0) Flow rate: 0.4 ml/min Temperature: 40 C Detection: UV260nm 1; icotinic Acid (0.4mg/ml) 2; icotinamide (0.6mg/ml) Inj. Vol: 0.5μl Thiamine Pyrophosphate, Thiamine Glycosides with identical aglycones but different glycosyl groups can also be separated. UDP Glycosides Arbutin and Hydroquinone - P - 2; ADP 3; AMP H 2 H H 2 H H 2 H H H H H H 2 1; icotinic Acid 2; icotinamide CSMCRE Application Data CSMCRE CSMCRE 2.6C PBr Column: CSMCRE 2.6PBr Column size: 2.1mmI.D.-150mm Mobile phase: 100mmol/l Phosphate buffer(ph2.5) Flow rate: 0.4 ml/min Temperature: 40 C Detection: UV245nm CSMCRE Application Data Column: CSMCRE 2.6PBr Column size: 2.1mmI.D.-150mm Mobile phase: 100mmol/l Phosphate buffer(ph7.0) Flow rate: 0.4 ml/min Temperature: 40 C Detection: UV260nm 1; UDP (0.8mg/ml) 2; UDP-Galactose (0.8mg/ml) 3; UDP-Glucose (0.8mg/ml) Inj. Vol: 0.5μl P - H H H P P - - H H H H H H H P P - - H H 1; UDP 2; UDP-Galactose 3; UDP-Glucose H H H H H 1; Thiamine Pyrophosphate (0.4mg/ml) 2; Thiamine (0.4mg/ml) Inj. Vol: 0.5μl H 3C H 2 + H 3C S H H P P H H 3C H 2 + H 3C 1; Thiamine Pyrophosphate 2; Thiamine CSMCRE Application Data S H Column: CSMCRE 2.6PBr Column size: 2.1mmI.D.-100mm Mobile phase: Acetonitrile/ 20mmol/l Phosphate buffer(ph2.5) = 5/95 Flow rate: 0.4 ml/min Temperature: 40 C Detection: UV290nm 1; Arbutin (1.33mg/ml) 2; Hydroquinone (0.67mg/ml) Inj. Vol: 0.75μl H H H H H H H 1; Arbutin 2; Hydroquinone I. HPLC Columns II. SFC Columns III. Preparative Packing Materials IV. Related Products rdering Information Analytical Columns (Particle Size: 2.6 m) CSMCRE 2.6PBr Packed Column 2.1 x x x x x x x x x x CSMCRE s connector is the same type as Waters UPLC columns. For UHPLC-compatible pre lters, refer to page x x x x x x

14 I. HPLC Columns (2) Instrument Settings and Compatibility When using with a conventional (non-uhplc) instrument CSMCRE columns are designed for use with UHPLC instruments. In addition, due to their low backpressure, they can be used with conventional instruments. However, it is necessary to change the following settings. Detector Response Time Because UHPLC analyses are done at high flow rates, a slow response time can adversely affect peak shape. We recommend setting the response time to 0.1 sec or less. CSMCRE Application Data II. SFC Columns Column: CSMCRE 2.6C 18 Column size: Mobile phase: Acetonitrile/ Water = 70 /30 Flow rate: 3.0mmI.D.-75mm 2.1mmI.D.-50mm 1.0 ml/min 0.4ml/min Temperature: 40 C UV254nm 1; Benzene 2; Toluene 3; Ethylbenzene 4; Propylbenzene 5; Butylbenzene 6; Amylbenzene (2.0mg/ml) (2.0mg/ml) (2.0mg/ml) (2.0mg/ml) (2.0mg/ml) (2.0mg/ml) 3.0mmI.D.-75mm (0.02sec) (1.0sec) 2.1mmI.D.-75mm (0.02sec) (1.0sec) Inj.Vol.: 3.0mmI.D.-75mm 2.1mmI.D.-50mm 1.0μl 0.5μl III. Preparative Packing Materials IV. Related Products ther Instrument Parameters UHPLC is more vulnerable to the effects of dead volume than conventional chromatography. When using a 2.1 mm I.D. column, please use a semi-micro detector cell, injector, and piping (0.1mm). CSMCRE Application Data Column: Column size: Mobile phase: Acetonitrile/ Water = 70/30 Flow rate: 3.0mmI.D.-75mm 1.0 ml/min Fittings and Adapters 3.0mmI.D.-75mm 2.1mmI.D.-75mm CSMCRE 2.6C 18 Semi-micro Standard Semi-micro Standard 2.1mmI.D.-50mm Temperature: 40 C Detection: UV254nm 0.4ml/min 1; Benzene (2.0mg/ml) 2; Toluene (2.0mg/ml) 3; Ethylbenzene (2.0mg/ml) 4; Propylbenzene (2.0mg/ml) 5; Butylbenzene (2.0mg/ml) 6; Amylbenzene (2.0mg/ml) Inj.Vol.: 3.0mmI.D.-75mm 1.0μl 2.1mmI.D.-50mm 0.5μl CSMCRE columns use the same connectors as Waters UPLC (UHPLC) columns. This is different from our conventional CSMSIL columns, which use the conventional Waters HPLC-compatible connectors. (UPLC is a registered trademark of Waters Corporation.) Connection Type Instrument HPLC(CSMSIL) Column UHPLC (CSMCRE) HPLC o adapter required Adapter required UHPLC Adapter required o adapter required HPLC: Conventional Waters-compatible connector UHPLC: Waters UPLC -compatible connector HPLC UHPLC Connector Shape approx. 3.3 mm approx. 2.3 mm The length of tubing that extends from the ferrule differs from HPLC to UHPLC. 12

15 o adapter needed; just connect as-is. An adapter or movable (high-pressure) tting is required to connect the ttings to the column. See the examples for different ttings below. The column can be connected by using an adapter (SUS union tubing xed to UHPLC length). PEEK ttings do not x the length of tubing at the end, so they can be used with both types of column. However, please be cautious of their pressure tolerance. approx. 3.3 mm Common PEEK fitting (Max. pressure: 28MPa) R High-pressure fitting rdering Information Adapter List approx. 2.3 mm In Adapter In FLW tubing fixed at 2.3 mm FLW ut ut approx. 2.3 mm In ut Adapter FLW Common PEEK fitting (Max. pressure: 28MPa) R High-pressure fitting tubing fixed at 2.3 mm approx. 3.3 mm Product ame Description PKG Size Low & Zero Dead Volume Union Material: SUS Bore diameter: 0.35 mm P PKG CSMSIL Column Connecting Tube (0.1 mm I.D.) I.D.: 0.1 mm PKG CSMSIL Column Connecting Tube (0.25 mm I.D.) I.D.: 0.25 mm PKG I. HPLC Columns II. SFC Columns III. Preparative Packing Materials IV. Related Products UHPLC-compatible pre lter Product ame In ut Contents PKG Size U-Fil UHPLC-compatible pre lter U-Fil replacement lter - - UHPLC UHPLC Filter: 0.5 m SET HPLC UHPLC Tubing connecting diameter:1/ SET Filter: 0.5 m Material: SUS316L units / PKG 13

16 I. HPLC Columns 2. HPLC Columns (1) Reversed Phase Columns C18 (DS) Series Speci cations Packing Material C18-MS-II C18-AR-II C18-PAQ C18-EB Silica Gel High purity porous spherical silica Average Particle Size 2.5, 3, 5, 15 m 3, 5, 15 m 5, 15 m 3 m Average Pore Size approx. 120 Speci c Surface Area approx. 300 m 2 /g II. SFC Columns Bonded Phase Structure III. Preparative Packing Materials IV. Related Products Bonded Phase ctadecyl group Bonding Type Monomeric Polymeric Monomeric Main Interaction Hydrophobic interaction End-Capping Treatment ear-perfect treatment Carbon Content approx. 16% approx. 17% approx. 11% approx. 14.5% Usable ph Range 2~10* 1.5~7.5* 2~ * Multi-purpose C18 Column Features strong acid resistance. Good for hydrophilic compounds. Good for basic compounds Features Good for acidic compounds and Stable performance under peptides. 100% aqueous conditions. *ptimal ph range of silica-based columns is between 2 and 7.5. Extreme ph may signi cantly decrease column lifetime. Difference in Separation Properties (5 m) H3C Si CH3 CSMSIL 5C18-AR-II retains planar compouds (such as triphenylene) longer compared to CSMSIL 5C18-MS-II. CSMSIL 5C18-PAQ has shorter retention time, and retains polar compounds (Such as valerophenone, n-butyl benzoate) longer. Difference in Separation Properties Column: Column size: 4.6mmI.D.-150mm Mobile phase: Methanol/H 2 = 80/20 Temperature: 30 C Detection: UV254nm 1; Valerophenone 2; n-butyl Benzoate 3; n-butylbenzene 4; o-terphenyl 5; Amylbenzene 6; Triphenylene (0.17 g) (0.17 g) (8.0 g) (0.17 g) (8.0 g) (0.02 g) Si 5C 18 -MS-II 5C 18 -AR-II 5C 18 -PAQ H H3C Si CH3 AP

17 CSMSIL C18-MS-II First-choice column of our DS series Multi-purpose C18 column High reproducibility A wide range of applications < Suitable Samples > Low-M.W. Compounds Separation Property The CSMSIL 5C18-MS-II is a well-balanced column with better basic performance, such as sharper peaks for basic compounds and chelating compounds, strong hydrophobic interaction, low analytical pressure, and high theoretical plate number. CSMSIL 5C18-MS-II is the rst-choice column for reversed-phase chromatography. Separation Property Column: 5C 18 -MS-II Column size: 4.6mmI.D.-150mm Mobile phase: Methanol/ H 2 = 60/40 Detection: UV254nm 1; Acetophenone (0.05 g) 2; Methyl Benzoate (0.5 g) 3; Benzene (2.0 g) 4; Toluene (2.0 g) AP-0421 Table. Comparison of hydrophobic interaction, analytical pressure, and theoretical plate number Column Analysis of Basic Compounds and Metal Coordination Compounds Column Characteristics Dipole-Dipole Interaction Hydrophobic Interaction (Toluene/Benzene) Hydrophobic Interaction Pressure (MPa) Molecular Shape Selectivity π-π Interaction 5C18-MS-II Theoretical Plate umber (Toluene) CSMSIL 5C18-MS-II Company A C Company B C Company C C Company D C The CSMSIL 5C18-MS-II column, taking advantage of a new end-capping treatment, can replace the original CSMSIL C18 (DS) column. A new end-capping treatment with polar groups for shielding effect has signi cantly improved peak shape for basic compounds. Ultra pure silica gel with low trace-metal content is used for CSMSIL columns; thus the columns provide excellent peak shapes for chelating compounds. Basic Compounds Column: 5C 18 -MS-II Column size: 4.6mmI.D.-150mm Mobile phase: Methanol/ 20mmol/l Phosphate buffer(ph7) = 20/80 Detection: UV254nm 1; Procainamide (0.38 g) 2; -Acetylprocainamide (0.25 g) 3; Benzylalcohol (5.63 g) Dispersion Force Metal Coordination Compounds Column: 5C 18 -MS-II Column size: 4.6mmI.D.-150mm Mobile phase: Acetonitrile / 20mmol/l Phosphoric Acid = 5/95 Detection: UV240nm xine-copper I. HPLC Columns II. SFC Columns III. Preparative Packing Materials IV. Related Products AP-1010 AP-0143 High Reproducibility The strict quality control system of acalai Tesque ensures the quality of the silica gel and bonding and end capping process, reducing variation between lots. We support customers with an individual Inspection Report which accompanies each and every CSMSIL, CSMCRE and CSMGEL packed column (except guard columns) and an additional Certi cate of Analysis for the CSMSIL 5C18-MS-II (4.6 mm I.D. x 150 mm and 4.6 mm I.D. x 250 mm). 15

18 A Wide Range of Applications I. HPLC Columns A wide selection of applications, e.g. drug molecules, is available to achieve appropriate separation parameters for target samples. Parasympatholytic Agents Analgesic Antipyretic Drugs CSMSIL Application Data Column: 5C 18 -MS-II Column size: 4.6mmI.D.-150mm Mobile phase: Methanol/ 20mmol/l Phosphate buffer(ph7) = 30/70 Detection: UV254nm, 0.16AUFS II. SFC Columns rdering Information 1; p-acetamidophenol 2Ati 2; Antipyrinei 3; 4-Aminoantipyrine 4; Phenacetin AP-0070 III. Preparative Packing Materials IV. Related Products Analytical / Preparative Columns (Particle Size: 5 m) CSMSIL 5C18-MS-II Packed Column 1.0 x x x x x x x x x x x x Analytical Columns (Particle Size: 2.5 m) CSMSIL 2.5C18-MS-II Packed Column 4.6 x x 150* x lots set x 250* x x x x x x x x CSMSIL 5C18-MS-II Guard Column 4.6 x x 10 Cartridge** x x x x * Validated columns ** 2 cartridges included. Guard cartridge holder required; refer to page 71. Preparative Columns (Particle Size: 15 m) Fast LC Column (Particle Size: 3 m) CSMSIL 15C18-MS-II Packed Column CSMSIL 15C18-MS-II Guard Column CSMSIL 3C18-MS-II Packed Column 28 x x x x x x x x x x x x x x x

19 CSMSIL C18-AR-II Features strong acid resistance < Suitable Samples > Peptides, acidic compounds, etc. Acid Resistance The CSMSIL 5C18-AR-II packed column features a polymeric type of C18 reversed phase material. The acidic resistance of CSMSIL 5C18-AR-II is much improved compared with commercially available monomeric type octadecyl stationary phases. It retains high performance even with acidic mobile phases commonly used to separate acidic compounds and peptides k'(%) 70 Applications Peptides 60 CSMSIL 5C18-AR-II Conventional monomeric C Time (hour) CSMSIL Application Data Column: 5C 18 -AR-II Column size: 4.6mmI.D.-150mm Mobile phase: A; 0.05%TFA-H 2 B; 0.05%TFA-Acetonitrile B conc % 20min Linear gradient Detection: UV220nm 1; xytocin (0.8 g) 2; Angiotensin II(Human) (0.8 g) 3; Angiotensin I(Human) (0.8 g) 4; Substance P (0.8 g) AP-0351 Column Characteristics Dispersion Force Dipole-Dipole Interaction Hydrophobic Interaction 5C18-AR-II Molecular Shape Selectivity π-π Interaction 5C18-MS-II Decomposition test in 0.1% Tri uoroacetic acid solution at 60 o C. Capacity factor (k ) = aphthalene, Mobile phase: Methanol / H2=70/30 rganic Acids CSMSIL Application Data Column: 5C 18 -AR-II Column size: 4.6mmI.D.-150mm Mobile phase: Methanol/ 20mmol/l Phosphoric Acid = 20/80 Detection: UV254nm, 0.5AUFS 1; Gallic Acid (0.63 g) 2; Protocatechuic t Acid (0.63 g) 3; Gentisic Acid (0.63 g) 4; Phthalic Acid (0.63 g) AP-0159 I. HPLC Columns II. SFC Columns III. Preparative Packing Materials IV. Related Products Salicylic Acid Esters Parabens CSMSIL Application Data Column: 5C 18 -AR-II Column size: 4.6mmI.D.-150mm Mobile phase: Acetonitrile/ H 2 = 50/50 Detection: UV254nm, 0.12AUFS 1; Methyl p-hydroxybenzoate (0.125 g) 2; Ethyl p-hydroxybenzoate (0.125 g) 3; n-propyl P lp-hydroxybenzoate (0.125 g) 4; Butyl p-hydroxybenzoate (0.125 g) AP

20 LC/MS Applications I. HPLC Columns HPLC 5C18-AR- 2.0 mm l.d. x 150 mm UV Aconitine MW Impurity may lack this part Enables simultaneous analysis of multiple wavelength spectrum II. SFC Columns MS MS III. Preparative Packing Materials IV. Related Products Impurity (MW 586 rdering Information Analytical / Preparative Columns (Particle Size: 5 m) CSMSIL 5C18-AR-II Packed Column 1.0 x x x x x x x x x x x x bjective substance Aconitine (MW 646) 4.6 x x 150* x lots set x 250* x x x x x x x x CSMSIL 5C18-AR-II Guard Column 4.6 x x 10 Cartridge** x x x x * Validated columns ** 2 cartridges included. Guard cartridge holder required; refer to page 71. Preparative Columns (Particle Size: 15 m) CSMSIL 15C18-AR-II Packed Column 28 x x x CSMSIL 15C18-AR-II Guard Column 28 x x Fast LC Column (Particle Size: 3 m) CSMSIL 3C18-AR-II Packed Column 2.0 x x x x

21 CSMSIL C18-PAQ Compatible with 100% water based mobile phase < Suitable Samples > Hydrophilic compounds rganic acids, nucleic acid bases, etc. Stable Performance Stable performance under 100% aqueous conditions tr(%) (0min taken as 100) Applications rganic Acids Mobile phase flow stopped Analysis resumed 92 CSMSIL 5C18-PAQ Conventional C min CSMSIL Application Data Column: 5C 18 -PAQ Column size: 4.6mmI.D.-250mm Mobile phase: 20mmol/l Phosphate buffer(ph2.5) Detection: UV210nm 1; Glycolic Acid (6.3 g) 2; Tartaric Acid (4.0 g) 3; L (-)-Malic ( ) Mli Acid Aid (6.4 g) 4; Lactic Acid (13 g) 5; Acetic Acid (13 g) 6; Citric Acid(6.3 g) 7; Succinic Acid (13 g) 8; Maleic Acid (0.06 g) 9; Propionic Acid (13 g) 10; Fumaric Acid (0.03 g) dtps CSMSIL Application Data AP-1058 Column Characteristics Dispersion Force Dipole-Dipole Interaction Hydrophobic Interaction 5C18-PAQ Molecular Shape Selectivity π-π Interaction 5C18-MS-II The gure shows the change of retention time for thymine with 100% aqueous mobile phase (20 mmol/l phosphate buffer, ph 7). The sample was analyzed 4 times (1 hour). Flow of mobile phase was then stopped for 1 hour. The sample was analyzed under the same conditions again after 1 hour. The conventional C18 column showed change of retention time, but CSMSIL 5C18-PAQ maintained stable retention time. ucleobases and ucleosides CSMSIL Application Data Column: 5C 18 -PAQ Column size: 4.6mmI.D.-150mm Mobile phase: 20mmol/l Phosphate buffer(ph7) Detection: UV260nm 1; Cytosine (0.16 g) 2; Uracil (0.16 g) 3; Cytidine (0.32 g) 4; Uridine (0.24 g) 5; Guanine (0.16 g) 6; Thymine (0.16 g) 7; Adenine (0.08 g) 8; Guanosine (0.24 g) 9; Thymidine(0.24 g) 10; Adenosine (0.16 g) Catecholamines CSMSIL Application Data AP-0375 I. HPLC Columns II. SFC Columns III. Preparative Packing Materials IV. Related Products Column: 5C 18 -PAQ Column size: 4.6mmI.D.-250mm Mobile phase: 20mmol/l Phosphate buffer(ph7) Detection: UV260nm, 0.16AUFS 1; 2 -Deoxycytidine 5 -Triphosphate [dctp] (1mmol/l) 2; 2 -Deoxythymidine 5 -Triphosphate [dttp] (1mmol/l) 3; 2 -Deoxyguanosine 5 -Triphosphate [dgtp] (1mmol/l) 4; 2 -Deoxyadenosine 5 -Triphosphate [datp] (1mmol/l) Injection 0.5 l AP-0371 Column: 5C 18 -PAQ Column size: 4.6mmI.D.-250mm Mobile phase: 20mmol/l Phosphate buffer(ph7.0) Detection: UV270nm 1; L-oradrenaline (0.38 g) 2; ( )-Epinephrine Ei hi (0.38 g) 3; DL-3-Methoxy -4-hydroxymandelic Acid (0.56 g) 4; L-DPA (0.56 g) 5; Dopamine (1.13 g) 6; Homovanillic Acid (1.13 g) AP

22 Applications Ascorbic Acid 2-Phenylethyl Glycosides I. HPLC Columns CSMSIL Application Data Column: 5C 18 -PAQ Column size: 4.6mmI.D.-250mm Mobile phase: 20mmol/l Phosphoric Acid Detection: UV245nm, 0.16AUFS CSMSIL Application Data Column: 5C 18 -PAQ Column size: 4.6mmI.D.-150mm Mobile phase: Acetonitrile/ Methanol/ H 2 = 8/4/888 Temperature: 30 C Detection: UV210nm (+)-Ascorbic Acid [Vitamin C] (0.1 g) 1; 2-Phenylethyl- -melibioside 2; 2-Phenylethyl- -gentiobioside 3; 2-Phenylethyl- -lactoside 4; 2-Phenylethyl- -cellobioside 5; 2-Phenylethyl- -maltoside II. SFC Columns III. Preparative Packing Materials IV. Related Products rdering Information Analytical / Preparative Columns (Particle Size: 5 m) CSMSIL 5C18-PAQ Packed Column 1.0 x x x x x x x x x x x x Preparative Columns (Particle Size: 15 m) CSMSIL 15C18-PAQ Packed Column 28 x x x AP x x x x x x x x x x x CSMSIL 15C18-PAQ Guard Column 28 x x Data courtesy of Dr. K. Sakata, Dr. B. Shimizu, Institute for Chemical Research, Kyoto University CSMSIL 5C18-PAQ Guard Column 4.6 x x x x x

23 CSMSIL C18-EB 3 m C18 column with reduced tailing and high resolution Usable with simple mobile phases < Suitable Samples > For quality control of drugs Compounds that induce peak tailing, such as basic compounds Analysis of Basic Compounds CSMSIL 3C18-EB uses a new end-capping method to reduce the number of residual silanol groups, which can cause peak tailing with basic compounds. CSMSIL Application Data Column: Column size: 4.6mmI.D.-75mm Mobile phase: Acetonitrile/ 20mmol/l Phosphate buffer(ph7.0) = 60/40 Temperature: 40 C Detection: UV254nm 1; Amitriptyline (0.2mg/ml) 2; Propylbenzene (I.S.) (2.0mg/ml) Inj. Vol: 1.0μl Amitriptyline CSMSIL 3C 18 -EB Competitor A (3 m, C 18 ) Analysis of Acidic Compounds and Metal Coordination Compouds Column Characteristics Competitor B (3 m, C 18 ) Dipole-Dipole Interaction Hydrophobic Interaction CSMSIL 3C18-EB utilizes a new end-capping method and high-purity silica gel to reduce tailing with metal coordination compounds. Acidic Compounds Metal Coordination Compounds CSMSIL Application Data Column: 3C 18 -EB Column size: 4.6mmI.D.-75mm Mobile phase: Acetonitrile/ 20mmol/l Phosphate buffer(ph2.5) = 30/70 Temperature: 40 C Detection: UV254nm 1; Salicylic Acid (1.0mg/ml) 2; Ethyl p-hydroxybenzoate (I.S.)(0.05mg/ml) Inj. Vol: 1.0μl H H Salicylic Acid Dispersion Force CSMSIL Application Data Column: 3C 18 -EB Column size: 4.6mmI.D.-75mm Mobile phase: Acetonitrile/ 20mmol/l Phosphoric Acid = 10/90 Temperature: 40 C Detection: UV254nm 1; xine-copper (0.03mg/ml) 2; Caffeine (I.S.) (0.3mg/ml) Inj. Vol: 2.0μl Cu xine-copper 5C18-EB Competitor C (3 m, C 18 ) Molecular Shape Selectivity π-π Interaction 5C18-MS-II I. HPLC Columns II. SFC Columns III. Preparative Packing Materials IV. Related Products rdering Information Analytical Columns (Particle Size: 3 m) CSMSIL 3C18-EB Packed Column CSMSIL 3C18-EB Guard Column Column Size Column Size Column Size I.D. x Length (mm) 2.0 x x x 10 Cartrige** x x x x x x 10 Cartrige** x x 75* * Validated columns 2.0 x x 100* ** 2 cartridges included. Guard cartridge 3.0 x x 150* holder required; refer to page x x x

24 Reversed Phase Specialty Columns I. HPLC Columns Speci cations Packing Material Cholester PBr AP PYE PE Silica Gel High purity porous spherical silica Average Particle Size 2.5, 5 m 5 m 2.5, 5 m 5 m Average Pore Size approx. 120 Speci c Surface Area approx. 300 m 2 /g Br H3C Br Br II. SFC Columns III. Preparative Packing Materials Bonded Phase Structure Bonded Phase Bonding Type Main Interaction Cholesteryl group Hydrophobic interaction Molecular shape selectivity Pentabromobenzyl group Hydrophobic interaction Dispersion force aphtylethyl group Monomeric Hydrophobic interaction - interaction Pyrenylethyl group Hydrophobic interaction - interaction Dispersion force Molecular shape selectivity itrophenylethyl group Hydrophobic interaction - interaction Dipole-dipole interaction End-Capping Treatment ear-perfect treatment Carbon Content approx. 20% approx. 8% approx. 11% approx. 18% approx. 9% ph Range Features H3C H3C Usable under the same conditions as C18 High molecular shape selectivity Separate hydrophilic compounds under reversed-phase conditions Selectivity for positional isomers of dinitrobenzene Si CH3 Br H3C Si CH3 Br Stronger - interaction than phenyl columns Very strong - interaction Strong dipole-dipole interaction Different stationary phase exhibits different selectivity due to the use of forces that C18 (hydrophobic interaction) does not have. By using these columns, you can achieve separation that cannot be done using only C18. H3C Si CH3 H3C Si CH3 H3C Si 2 CH3 IV. Related Products C18-MS-II Cholester PBr AP PE Column size: 4.6mmI.D.-150mm Mobile phase: Methanol/ H 2 = 50/50 Temperature: 30 C Detection: UV254nm o-dinitrobenzene m-dinitrobenzene p-dinitrobenzene Benzene (I.S.) Bezene(I.S.) o- m- p- 22

25 CSMSIL Cholester Cholesterol-bonded stationary phase Increased stereoselectivity and improved resolution for geometric isomers Usable under the same conditions as C18 < Suitable Samples > atural compouds, polyphenols, catechins, fat-soluble vitamins and avones Molecular Shape Selectivity The stationary phase of Cholester has a very rigid structure and can distinguish different molecular shapes. Cholester retains planar triphenylene longer than non-planar o-terphenyl. Comparison of Molecular Shape Selectivity Column: Column size: 4.6mmI.D.-150mm Mobile phase: Methanol/H 2 = 90/10 Detection: UV254nm 1; o-terphenyl (0.1 g) 2; Triphenylene (0.01 g) o-terphenyl() Hydrophobic Interaction Triphenylene(T) 5C 18 -MS-II Column Characteristics Dipole-Dipole Interaction Hydrophobic Interaction The below gure shows the comparison of hydrophobic interactions with competitor's C18 columns. Cholester provides about the same hydrophobicity as alkyl group bonded types (C18, C30). It is not necessary to change the analytical conditions when replacing C18 or C30 columns with Cholester. Comparison of Hydrophobic Interaction Column: Column size: 4.6mmI.D.-150mm Mobile phase: Methanol/H 2 = 80/20 Detection: UV254nm 1; Benzene (1.67 g) 2; Toluene (1.67 g) 3; Ethylbenzene (1.67 g) 4; n-propylbenzene (1.67 g) 5; n-butylbenzene (1.67 g) 6; Amylbenzene (1.67 g) (CH 2) n-h n=0-5 5C 18 -MS-II Dispersion Force Cholester Cholester Cholester Molecular Shape Selectivity π-π Interaction AP C18-MS-II I. HPLC Columns II. SFC Columns III. Preparative Packing Materials IV. Related Products AP-1018 Applications Catechins CSMSIL Application Data 5C 18 -MS-II Cholester Column: Column size: 4.6mmI.D.-150mm Mobile phase: A: Acetonitrile/ 20mmol/l Phosphate buffer(ph2.5) = 10/90 B: Acetonitrile/ 20mmol/l Phosphate buffer(ph2.5) = 30/70 B conc % 20min Linear gradient Detection: UV280nm 1; (-) Gallocatechin (-) GC ) (0.80mg/ml) 2; Caffeine (0.08mg/ml) 3; (-) Epigallocatechin (-) EGC (0.80mg/ml) 4; (-) Catechin (-) C (0.40mg/ml) 5; (-) Epicatechin (-) EC (0.40mg/ml) 6; (-) Epigallocatechin Gallate (-) EGCg ) (0.20mg/ml) 7; (-) Gallocatechin Gallate (-) GCg (0.40mg/ml) 8; (-) Epicatechin Gallate (-) ECg (0.20mg/ml) 9; (-) Catechin Gallate (-) Cg (0.20mg/ml) Injection Vol. 1.0 l AP

26 I. HPLC Columns Applications (Continued) 2.5 m particles yield better performance and shorter analysis time compared to 5 m particles. Saikosaponins CSMSIL Application Data Mobile phase: Acetonitrile/ 0.05%aH 2 P 4 aq. = 3/5 Flow rate: 0.7 ml/min Temperature: 50 C Detection: UV206nm 1; Saikosaponin c 2; Saikosaponin h 3; Saikosaponin a 4; Saikosaponin b2 5; Saikosaponin b1 6; Saikosaponin d Injection Vol. 1.0μl Company C 1.7μmC 18 (2.1mmI.D.-50mm) CSMSIL 2.5Cholester (2.0mmI.D.-50mm) II. SFC Columns Water-Soluble Vitamins CSMSIL Application Data Company C C μm Company D 2.6u C 18 (Core-Shell) CSMSIL 2.5C 18 -MS-II High resolution CSMSIL 2.5Cholester III. Preparative Packing Materials Column: Column size: Mobile phase: A; 0.1% TFA-Methanol/ H 2 = 90/10 B; 0.1% TFA-Methanol B conc. 0%(0-5min)-100%(10min)-100%(15min) Flow rate: 0.4 ml/min Temperature: 40 C Detection: UV280nm 1; Vitamin A Acetate, all trans (0.06mg/ml) 2; Vitamin D 2 [Calciferol] (0.30mg/ml) 3; Vitamin D 3 (0.06mg/ml) 4; Vitamin E Succinate [D-α-Tocopherol Succinate] (0.60mg/ml) 5; Vitamin K 2 (0.18mg/ml) 6; Vitamin E [DL-α-Tocopherol] (0.60mg/ml) 7; Vitamin E Acetate [DL-α-Tocopherol Acetate] (0.60mg/ml) 8; Vitamin K 1 (0.18mg/ml) 9; Vitamin A Palmitate (0.18mg/ml) Inj. Vol: 2.5μl (2.1mmI.D.-50mm) (2.1mmI.D.-50mm) (2.0mmI.D.-50mm) (2.0mmI.D.-50mm) IV. Related Products rdering Information Analytical / Preparative Columns (Particle Size: 5 m) CSMSIL Cholester Packed Column 1.0 x x x x x x x x x x 150* x lots set* x 250* x x x x x * Validated Columns CSMSIL Cholester Guard Column 4.6 x x x x x Analytical (Particle Size: 2.5 m) CSMSIL 2.5Cholester Packed Column 2.0 x x x x x x

27 CSMSIL PBr Pentabromobenzyl-bonded stationary phase Separate hydrophilic compounds in reversed-phase conditions < Suitable Samples > Hydrophilic compounds ucleotides, peptides, catecholamines and oligosaccharides Comparison with C18 CSMSIL PBr retains hydrophilic compounds stronger than C18 columns under the same reversed-phase conditions. CSMSIL Application Data Column: Column size: 4.6mmI.D.-250mm Mobile phase: 100mmol/l Phosphate buffer(ph7.0) Temperature: 30 C Detection: UV260nm 1; Uridine-5'-diphosphate (0.8mg/ml) 2; Uridine-5 -diphosphogalactose (0.8mg/ml) 3; Uridine-5 -diphosphoglucose (0.8mg/ml) Inj.Vol.: 1.0μl H H H H Comparison with HILIC P P H H Uridine-5'-diphosphoglucose H H H Column Characteristics Dipole-Dipole Interaction Hydrophobic Interaction HILIC is widely recognized as a method for separating hydrophilic compounds. However, because it differs from the commonly used reversed-phase mode, setting mobile phase conditions can be difficult. In addition, the use of acetonitrile in high concentration can cause problems with peak shape when using water as a sample solvent. CSMSIL PBr can retain hydrophilic compounds under reversed-phase conditions, and maintains good peak shape even when injecting large amounts of water. CSMSIL Application Data Column: HILIC Column size: 4.6mmI.D.-150mm Mobile phase: Acetonitrile/ H 2 = 90/10 Temperature: 30 C Detection: UV260nm 1; Uracil (0.1mg/ml) 2; Uridine (0.2mg/ml) Sam. Solution H 2 H H H H H H H H H 1; Uracil 2; Uridine H Undiluted sol. 1μl Injection 1/20 dilute sol. 20μl Injection 5C 18 -PAQ Column: PBr Column size: 4.6mmI.D.- 150mm Mobile phase: H 2 Temperature: 30 C Detection: UV260nm 1; Uracil (0.1mg/ml) 2; Uridine (0.2mg/ml) Sam. Solution H 2 PBr Dispersion Force Undiluted sol. 1μl Injection PBr Molecular Shape Selectivity π-π Interaction AP /20 dilute sol. 20μl Injection 5C18-MS-II I. HPLC Columns II. SFC Columns III. Preparative Packing Materials IV. Related Products rdering Information Analytical / Preparative Columns (Particle Size: 5 m) CSMSIL PBr Packed Column 2.0 x x x x x x x x x x x x x x x x x x x x x CSMSIL PBr Guard Column 4.6 x 10 Catrige* x x * 2 cartridges included. Guard cartridge holder required; refer to page

28 CSMSIL AP I. HPLC Columns aphthalene-bonded stationary phase Enhanced - interactions < Suitable Samples > Aromatic compounds and positional isomers Comparison of - Interactions Column Characteristics Dispersion Force Dipole-Dipole Interaction Hydrophobic Interaction πap Molecular Shape Selectivity π-π Interaction 5C18-MS-II C18-MS- CSMSIL AP shows stronger - interactions than phenyl columns. Its two fused aromatic rings retain nitrobenzene stronger than phenyl columns. II. SFC Columns Cholester PE-MS πap III. Preparative Packing Materials Applications α itrobenzene/benzene), 50%Methanol Figure. Comparison of - interaction Adrenal Cortical Hormones CSMSIL Application Data Column: Column size: 4.6mmI.D.-150mm Mobile phase: 5C 18 -MS-II Methanol/ H 2 = 50/50 5PE-MS Methanol/ H 2 = 50/50 AP Methanol/ H 2 = 60/40 Detection: UV254nm 5C 18 -MS-II 5PE-MS AP IV. Related Products 1; Prednisolone (0.33 g) 2; Hydrocortisone (0.33 g) 3; Cortisone (0.33 g) H H 1.Prednisolone H H H 2.Hydrocortisone H H 3.Cortisone H 2.5 m particles yield better performance and shorter analysis time compared to 5 m particles. Berberine AP-1023 CSMSIL Application Data CSMSIL 2.5C 18 -MS-II CSMSIL 2.5πAP Column: Column size: 3.0mmI.D.-75mm Mobile phase: Methanol/ 20mmol/l Phosphate buffer(ph2.5) 2.5C 18 -MS-II= 40/60 2.5πAP= 70/30 Temperature: 40 C Detection: UV254nm 1; Palmatine (0.125μg) 2; Berberine (0.125μg) AP

29 rdering Information Analytical / Preparative Columns (Particle Size: 5 m) 1.0 x x x x x x x x Analytical Columns (Particle Size: 2.5 m) 2.0 x x x x x x x x x x x x x x x x x x x I. HPLC Columns II. SFC Columns III. Preparative Packing Materials IV. Related Products 27

30 CSMSIL PYE I. HPLC Columns Pyrenylethyl-bonded stationary phase Stronger - interactions < Suitable Samples > Aromatic compounds, positional isomers, dioxins, and PCBs Comparison of - Interaction Column Characteristics Dispersion Force Dipole-Dipole Interaction Hydrophobic Interaction PYE Molecular Shape Selectivity π-π Interaction 5C18-MS-II C18-MS- CSMSIL PYE provides much stronger - interactions than AP page 26. II. SFC Columns Cholester PE-MS πap PYE α itrobenzene/benzene), 50%Methanol 3.0 Si H 3 C Si H 3 C Si H 3 C PE-MS AP PYE III. Preparative Packing Materials Applications Sterols Figure. Comparison of - interactions CSMSIL Application Data Column: Column size: 4.6mmI.D.-150mm Mobile phase: 5C 18 -MS-II Methanol/ H 2 = 98/2 AP Methanol/ H 2 = 90/10 5PYE Methanol/ H 2 = 95/5 Detection: UV210nm 5C 18 -MS-II AP 5PYE IV. Related Products 28 H Caution 1; Cholesterol (5.6 g) 2; Stigmasterol (5.9 g) 3; Campesterol 4; Sitosterol R R= 1. Methanol is the recommended mobile phase for CSMSIL PYE. Acetonitrile is not recommended because it has many electrons and interferes with - interactions between the sample and the stationary phase. 2. The stationary phase of CSMSIL PYE, pyrenylethyl group, has a large UV absorption. When the stationary phase detaches from silica gel and elutes, even a slight quantity can be detected and causes baseline noise. In such cases, wash the column with tetrahydrofuran. Detachment of a small amount of the stationary phase does not deteriorate a column s separation ability. 3. CSMSIL PYE column is not suitable for gradient analysis. rdering Information 1; H 3C 2; H 3C H 3C Analytical / Preparative Columns (Particle Size: 5 m) CSMSIL 5PYE Packed Column 1.0 x x x ; 4; H 3C 4.6 x x x x AP-1026 CSMSIL 5PYE Guard Column 4.6 x x x x

31 CSMSIL PE itrophenylethyl-bonded stationary phase Separation with dipole-dipole and - interactions < Suitable Samples > Isomers and nitro compounds Selectivity for Dipole-Dipole Interactions CSMSIL PE strongly retains 1,8-dinitronaphthalene because of the strong dipole formed by the two nitro groups positioned on the same side of naphthalene. Caution Comparison of Separation Property Column size: 4.6mmI.D.-150mm Mobile phase: PE Methanol / H 2 = 70/30 PYE Methanol / H 2 = 90/10 C 18 -MS-II Methanol / H 2 = 80/20 Detection: UV254nm 1; 1,8-Dinitronaphthalene 2; 1,5-Dinitronaphthalene 3; aphthalene 4; 1-Methylnaphthalene 5; 1,5-Dimethylnaphthalene Methanol is recommended as a mobile phase for CSMSIL PE. Acetonitrile is not recommended because it has many electrons and interferes with - interactions between the sample and the stationary phase. 2. The stationary phase of CSMSIL PE, nitrophenyl group, has a large UV absorption. When the stationary phase detaches from silica gel and elutes, even a slight quantity can be detected and causes baseline noise. In such a case, wash the column with tetrahydrofuran. Detachment of a small amount of the stationary phase does not deteriorate a column s separation ability. 3. CSMSIL PE column is not suitable for gradient analysis. rdering Information Analytical / Preparative Columns (Particle Size: 5 m) CSMSIL 5PE Packed Column 1.0 x x x PE 5PYE 4.6 x x x x Column Characteristics Dispersion Force Dipole-Dipole Interaction Hydrophobic Interaction 5C 18- MS-II PE Molecular Shape Selectivity π-π Interaction AP C18-MS-II CSMSIL 5PE Guard Column 4.6 x x x x I. HPLC Columns II. SFC Columns III. Preparative Packing Materials IV. Related Products 29

32 ther Reversed Phase Columns I. HPLC Columns Speci cations Packing Material PFP C-MS C22-AR-II C8-MS C4-MS TMS-MS PE-MS Silica Gel High purity porous spherical silica Average Particle Size 5 m Average Pore Size approx. 120 Speci c Surface Area approx. 300 m 2 /g II. SFC Columns III. Preparative Packing Materials Bonded Phase Structure Bonded Phase Penta uorophenyl group Cyanopropyl group Dococyl group ctyl group Butyl group Trimethyl group Bonding Type Monomeric Polymeric Monomeric Main Interaction F F H3C Hydrophobic interaction - interaction Dipole-dipole Hydrophobic interaction - interaction Hydrophobic interaction Phenylethyl group Hydrophobic interaction - interaction End-Capping Treatment ear-perfect treatment Carbon Content approx. 10% approx. 7% approx. 19% approx. 10% approx. 7% approx. 5% approx. 10% ph Range Difference in Separation Characteristics Si F F F CH3 H3C CSMSIL Application Data Si C CH3 Si H H3C Si CH3 5C 18 -MS-II 5C 22 -AR-II 5C 8 -MS H3C Si CH3 H3C H3C Si CH3 H3C Si CH3 IV. Related Products Column: Column size: 4.6mmI.D.-150mm Mobile phase: Methanol/ H 2 = 60/40 Temperature: 30 C Detection: UV254nm 1; Acetophenone (0.05μg) 2; Methyl Benzoate (0.5μg) 3; Benzene (2.0μg) 4; Toluene (2.0μg) 5C 4 -MS 5TMS-MS 5PFP 5PE-MS 5C-MS 30

33 CSMSIL PFP Penta uorophenyl-bonded stationary phase Alternative selectivity to C18 columns < Suitable Samples > Vitamin E, structural isomers and uorides Alternative Selectivity to C18 Columns CSMSIL PFP provides different selectivity from C18 Columns. Furthermore, it offers improved separation compared to other PFP columns. Applications CSMSIL Application Data Column: Column size: 4.6mmI.D.-150mm Mobile phase: Methanol/ H 2 = 40/60 Temperature: 30 C Detection: UV254nm o-cresol (3.0mg/ml) m-cresol (3.0mg/ml) p-cresol (3.0mg/ml) Inj.Vol.: 1.0μl Vitamin E rdering Information Analytical / Preparative Columns (Particle Size: 5 m) CSMSIL 5PFP Packed Column H H H o- m- p- CSMSIL Application Data Column: 5PFP Column size: 4.6mmI.D.-250mm Mobile phase: Methanol/ H 2 = 90/10 Temperature: 30 C Detection: UV295nm 1; -Tocotrienol 2; -Tocotrienol 3; -Tocotrienol 4; -Tocotrienol 5; -Tocopherol 6; -Tocopherol 7; -Tocopherol 8; -Tocopherol 2.0 x x x x x x x x x x x x Column Characteristics Dispersion Force Dipole-Dipole Interaction 5C 18 -MS-II Competitor PFP 5PFP Cresol Isomers CSMSIL Application Data Column: 5PFP Column size: 4.6mmI.D.-250mm Mobile phase: Methanol/ H 2 = 40/60 Temperature: 30 C Detection: UV254nm o-cresol (3.0mg/ml) m-cresol (3.0mg/ml) p-cresol (3.0mg/ml) Inj.Vol.: 1.0μl 10 x x x x x x x x x x x Hydrophobic Interaction H PFP Molecular Shape Selectivity π-π Interaction 5C18-MS-II CSMSIL 5PFP Guard Column 4.6 x 10 Cartrige* x x x * 2 cartridges included. Guard cartridge holder required; refer to page 71. H H o- m- p- 31 I. HPLC Columns II. SFC Columns III. Preparative Packing Materials IV. Related Products

34 I. HPLC Columns CSMSIL C-MS Cyanopropyl-bonded stationary phase Enables separation of different hydrophobic samples without using gradient < Suitable Samples > Mixtures of natural compounds Rapid Analysis Column Characteristics Gradient elution is commonly used for the samples containing both polar and non-polar compounds. However, gradient elution may cause reproducibility problems depending on the gradient mixer and pump, and needs equilibration time for each analysis. CSMSIL 5C-MS offers rapid analysis and great reproducibility using isocratic elution mode. Dispersion Force Dipole-Dipole Interaction Hydrophobic Interaction C-MS Molecular Shape Selectivity π-π Interaction 5C18-MS-II II. SFC Columns Application of Different Hydrophobic Samples Temperature: 30 C Detection: UV254nm 1; Theobromine 2; Theophylline 3; Caffeine 4; Benzene 5; Toluene 5C 18 -MS-II (4.6mmI.D.-150mm) Methanol/ H 2 = 20/80 5C-MS (4.6mmI.D.-150mm) Methanol/ H 2 = 20/80 5C 18 -MS-II (4.6mmI.D.-50mm) Methanol/ H 2 = 20/80 5 not elute 5C 18 -MS-II (4.6mmI.D.-150mm) Methanol/ H 2 = 50/50 Small hydrophobicity 1,2,3 not elute III. Preparative Packing Materials Large hydrophobicity Applications Acetoaminophen CSMSIL Application Data Column: Column size: 4.6mmI.D.-150mm 4,5 not elute 5C 18 -MS-II 5C-MS IV. Related Products Mobile phase: Methanol/ 50mmol/l KH 2 P 4 (ph4.7 with ah) = 20/80 Detection: UV225nm 1; p-aminophenol (0.2 g) 2; p-acetamidophenol (0.2 g) 3; 4 -Acetoxyacetanilide (0.2 g) AP-1075 rdering Information Analytical / Preparative Columns (Particle Size: 5 m) CSMSIL 5C-MS Packed Column 4.6 x x x x x x x x CSMSIL 5C-MS Guard Column 4.6 x x

35 CSMSIL C22-AR-II, C8-MS, C4-MS, TMS-MS, PE-MS rdering Information Analytical / Preparative Columns (Particle Size: 5 m) CSMSIL 5C22-AR-II Packed Column 4.6 x x x x CSMSIL 5C8-MS Packed Column 4.6 x x x x CSMSIL 5C4-MS Packed Column 4.6 x x x x CSMSIL 5TMS-MS Packed Column 4.6 x x x x CSMSIL 5PE-MS Packed Column 4.6 x x x x x x x x x x x x x x x x x x x x CSMSIL 5C22-AR-II Guard Column 4.6 x x CSMSIL 5C8-MS Guard Column 4.6 x x CSMSIL 5C4-MS Guard Column 4.6 x x CSMSIL 5TMS-MS Guard Column 4.6 x x CSMSIL 5PE-MS Guard Column 6.0 x x x x x x I. HPLC Columns II. SFC Columns III. Preparative Packing Materials IV. Related Products 33

36 I. HPLC Columns (2) ormal Phase Columns CSMSIL SL-II High purity silica gel (>99.99%) with special treatment Suitable for preparative separation Speci cations Packing Material SL-II Silica Gel High purity porous spherical silica Average Particle Size 3, 5, 15 m Average Pore Size approx. 120 Speci c Surface Area approx. 300 m 2 /g II. SFC Columns Features Comparison with ld Type High purity silica gel (>99.99%) with special treatment Suitable for preparative separation (higher resolution than medium-pressure or open chromatography) CSMSIL SL-II with high purity silica gel offers better peak shape for phenols with a simple mobile phase of ethanol or hexane. o acetic acid additives were required, unlike for the old type silica. III. Preparative Packing Materials Comparison with ld Type Column: Column size: 4.6mmI.D.-250mm Mobile phase: Ethanol/Hexane = 10/90 Detection: UV254nm 1; Benzoic Acid (4.0 g) 2; Salicylic Acid (6.0 g) 3; Salicylamide (4.0 g) 5SL-II 5SL (old type) IV. Related Products rdering Information Analytical / Preparative Columns (Particle Size: 5 m) CSMSIL 5SL-II Packed Column 4.6 x x x x CSMSIL 5SL-II Guard Column 6.0 x x x x x x x x x x Preparative Columns (Particle Size : 15 m) CSMSIL 15SL-II Packed Column CSMSIL15SL-II Guard Column 28 x x x x x Fast LC column (Particle Size: 3 m) CSMSIL 3SL-II Packed Column 4.6 x x x

37 (3) Hydrophilic Interaction Columns CSMSIL HILIC Triazole bonded stationary phase Enhanced hydrophilic interaction Unique anion-exchange mechanism < Suitable Samples > Hydrophilic compounds that would not be retained in reversed phase chromatography Melamine and water-soluble viatamins Speci cations Packing Material HILIC Silica Gel High purity porous spherical silica Average Particle Size 2.5, 5 m Average Pore Size approx. 120 Speci c Surface Area approx. 300 m 2 /g Bonded Phase Triazole Interaction Hydrophilic interaction, anion exchange Target Substance Hydrophilic compounds, acidic compounds Features Suitable for compounds not retained by C18 Comparison with C18 The hydrophilic interaction chromatography is a variation of normal phase chromatography where a polar stationary phase is used with a mobile phase which contains a high concentration of water-miscible organic solvent and a low concentration of aqueous eluent. The main retention mechanism is the partitioning of the polar analytes between the polar stationary and the non-polar mobile phase. As it is also called aqueous normal phase, the elution order is similar to that of normal phase and the sample elution is in the order of increasing hydrophilicity. Without using ion-pair reagent CSMSIL HILIC retains highly polar analytes that would not be retained in reversed phase chromatography. It also shows a weak anion-exchange mechanism with the positively charged stationary phase, thus acidic compounds are strongly retained. Comparison with C18 Column: Column size: 4.6mmI.D.-250mm Mobile phase: (HILIC) Acetonitrile/10mmol/l Ammonium Acetate = 60/40 (C 18 -PAQ) 20mmol/l Phosphate buffer(ph2.5) -(Ion pair)5mmol/l Sodium 1-ctanesulfonate Temperature: 30 C Detection: UV210nm 1; Glycine (5.0mg/ml) 2; Glycylglycine (0.125mg/ml) Inj.Vol: 2.0 l HILIC 5C 18 -PAQ without ion pair reagent 5C 18 -PAQ with ion pair reagent I. HPLC Columns II. SFC Columns III. Preparative Packing Materials IV. Related Products 35

38 Applications 2.5 m particles yield better performance and shorter analysis time compared to 5 m particles. I. HPLC Columns ucleotides CSMSIL Application Data Column: 2.5HILIC Column size: 2.0mmI.D.-50mm Mobile phase: Acetonitrile/ 20mmol/l Phosphate buffer(ph7.0) = 50/50 Flow rate: 0.4 ml/min Temperature: 40 C Detection: UV260nm Phosphorylated Peptide CSMSIL Application Data Column: 2.5HILIC Column size: 2.0mmI.D.-150mm Mobile phase: Acetonitrile/ 20mmol/l Phosphate buffer(ph7.0) = 70/30 Flow rate: 0.4 ml/min Temperature: 40 C Detection: UV220nm 1; Adenosine-5'-monophosphate (0.25mg/ml) 2; Adenosine-5'-diphosphate (0.50mg/ml) 3; Adenosine-5'-triphosphate (0.50mg/ml) Inj.Vol.: 0.5μl 1; Angiotensin II(Human) (0.5mg/ml) 2; [Tyr(P 3 H 2 ) 4 ]-Angiotensin II(Human) (0.5mg/ml) Inj.Vol.: 2.0μl II. SFC Columns Umami Components AP-1275 Melamine AP-1280 III. Preparative Packing Materials CSMSIL Application Data Column: 2.5HILIC Column size: 2.0mmI.D.-150mm Mobile phase: Acetonitrile/ 10mmol/l Phosphate buffer(ph7.0) = 50/50 Flow rate: 0.4 ml/min Temperature: 40 C Detection: UV210nm 1; L-Glutamic Acid (2.5mg/ml) 2; Succinic Acid (4.0mg/ml) 3; Inosine-5'-monophosphate (0.25mg/ml) 4; Guanosine-5'-monophosphate (0.25mg/ml) Inj.Vol.: 1.0μl rdering Information AP-1276 CSMSIL Application Data Column: 2.5HILIC Column size: 2.0mmI.D.-50mm Mobile phase: Acetonitrile/ 10mmol/l Ammonium Acetate = 80/20 Flow rate: 0.2 ml/min Temperature: 40 C Detection: ESI-MS 1; Melamine (1mg/L) 2; Ammeline (1mg/L) 3; Cyanuric Acid (1mg/L) 4; Ammelide (1mg/L) Inj.Vol.: 5.0μl ESI-MS, Positive, SIM ESI-MS, egative, SIM AP-1286 IV. Related Products Analytical / Preparative Columns (Particle Size: 5 m) CSMSIL HILIC Packed Column 1.0 x x x x x x x x x x 150* x lots set x 250* x x x * Validated Columns CSMSIL HILIC Guard Column 4.6 x x x x x Analytical Columns (Particle Size: 2.5 m) CSMSIL HILIC Packed Column 2.0 x x x x x x x x

39 (4) Mono- and ligosaccharide Analysis Columns Introduction Saccharides are not retained on standard C18 columns because of their low hydrophobicity. CSMSIL Sugar-D and H2- MS are speci cally designed for separation of saccharides. CSMSIL C18-PAQ is recommended for hydrophobic glycosides or saccharide derivatives. Speci cations Packing Material Sugar-D H2-MS Silica Gel High purity porous spherical silica Average Particle Size 5 m Average Pore Size approx. 120 Speci c Surface Area approx. 300 m 2 /g Bonded Phase Structure Bonded Phase Secondary/tertiary amine Aminopropyl group Bonding Type Polymeric Target Substances Monosaccharides, oligosaccharides End-Capping Treatment ear-perfect treatment Carbon Content approx. 4% Features First choice for saccharide analysis High durability Good quantitative analysis Different selectivity from Sugar-D Comparison of Retention The conventional aminopropyl column is slightly more retentive than Sugar-D. The retention time can be adjusted by increasing the concentration of acetonitrile in the mobile phase by 5%-10% as shown below. Comparison of Retention Column: Column size: 4.6mmI.D.-250mm Mobile phase: Detection: RI 1; D-(+)-Glucose 2; Maltose Sugar-D Acetonitrile / H 2 = 75/25 Acetonitrile / H 2 = 80/20 Si H2 H 5H 2 -MS Acetonitrile / H 2 = 75/25 I. HPLC Columns II. SFC Columns III. Preparative Packing Materials IV. Related Products 37

40 CSMSIL Sugar-D I. HPLC Columns ovel stationary phase for saccharides Superior durability compared to conventional amino columns Minimized undesirable adsorption Comparison of Durability The decrease of retention time was compared between CSMSIL Sugar-D and conventional aminopropyl bonded stationary phase with a severe 100% water eluent between tests. The capacity factor of Sugar-D did not decrease. II. SFC Columns III. Preparative Packing Materials Comparison of Adsorption Decomposition Condition Solution Water Flow Rate 1.0 ml/min Temperature Room Temperature Column 4.6 mm I.D. x 250 mm Mobile Phase Acetonitrile : Water = 70 : 30 Flow Rate 1.0 ml/min Temperature 30 o C Detection RI Sample Maltose Certain types of saccharides, such as arabinose or galactose, are partially or temporarily adsorbed on conventional aminopropyl stationary phases, causing tailing or no elution at all. CSMSIL Sugar-D provides superior separation and high recovery for these saccharides. Comparison of Adsorption Column: Column size: 4.6mmI.D.-250mm Mobile phase: Acetonitrile / H 2 = 70/30 Detection: RI Sugar-D 5H 2 -MS 1; D-(-)-Arabinose 2; D-(+)-Galactose 3; Maltitol IV. Related Products Applications Mono- and ligosaccharides Polyols CSMSIL Application Data CSMSIL Application Data Column: Sugar-D Column size: 4.6mmI.D.-250mm Mobile phase: Acetonitrile / H 2 = 75/25 Detection: RI Column: Sugar-D Column size: 4.6mmI.D.-250mm Mobile phase: Acetonitrile / H 2 = 75/25 Detection: RI 1; L-(+)-Rhamnose (10 g) 2; D-(-)-Fructose (10 g) 3; D-(+)-Glucose (10 g) 4; Sucrose (10 g) 5; Maltose (10 g) 6; D-(+)- Raffinose (10 g) 1; Glycerol (10 g) 2; -Erythritol E l Erythrite (10 g) 3; Xylitol (10 g) 4; Glucitol (10 g) 5; Maltitol (10 g) 6; Inositol (10 g) AP-0329 AP

41 Applications ligomaltoses ligofructoses CSMSIL Application Data Column: Sugar-D Column size: 4.6mmI.D.-250mm Mobile phase: Acetonitrile / H 2 = 65/35 Detection: RI 1; D-(+)-Glucose (10 g) 2; Maltose (10 g) 3; Maltotriose (10 g) 4; Maltotetraose (10 g) 5; Maltopentaose (10 g) 6; Maltohexaose (10 g) 7; Maltoheptaose (10 g) rdering Information Analytical / Preparative Columns (Particle Size : 5 m) CSMSIL Sugar-D Packed Column 2.0 x x x CSMSIL H2-MS CSMSIL Sugar-D Guard Column 4.6 x x x x x x x Aminopropyl-bonded stationary phase Different selectivity from Sugar-D Comparison of Adsorption AP-0331 H2-MS offers better separation than Sugar-D for some samples. Comparison of Separation Property Column: Column size: 4.6mmI.D.-250mm Mobile phase: (H 2 -MS) Acetonitrile / H 2 = 75/25 (Sugar-D) Acetonitrile / H 2 = 80/20 Detection: RI 1; Sucrose (10 g) 2; Maltose (10 g) 3; Lactose (10 g) 4; D-(+)-Trehalose (10 g) 5H 2 -MS CSMSIL Application Data Column: Sugar-D Column size: 4.6mmI.D.-250mm Mobile phase: Acetonitrile / H 2 = 70/30 Detection: RI Fructooligosaccharides (50 g) 1; 1-Kestose 2; ystose 3; 1-Fructofuranosyl-D-nystose Sugar-D AP-0320 I. HPLC Columns II. SFC Columns III. Preparative Packing Materials IV. Related Products rdering Information Analytical / Preparative Columns (Particle Size: 5 m) CSMSIL 5H2-MS Packed Column 4.6 x x x x CSMSIL 5H2-MS Guard Column 4.6 x x x

42 (5) Protein Separation Columns I. HPLC Columns Protein separation with HPLC < Separation Mode > < Packing Materials > Reversed Phase [Separation mechanism] Difference in hydrophobicity [Main applications] Protein analysis Purification of peptides *Proteins may become denatured. CSMSIL Protein-R Excellent separation High recovery rate utstanding stability at low ph Gel Filtration CSMSIL Diol Series II. SFC Columns Analysis / purification method of proteins [Separation mechanism] Size-based separation [Main applications] Purification of proteins Elimination of low-m.w.compounds Ion Exchange [Separation mechanism] Difference in surface charge [Main applications] Analysis and purification of proteins Ideal for the size-based separation of proteins and water-soluble polymers Reduce undesirable adsorption CSMGEL IEX Series Anion-exchange type, Cation-exchange type, Amphoteric ion-exchange type For Purification, for Ultra-fast analysis, for Precise analysis III. Preparative Packing Materials Reversed Phase Columns Hydrophobic Chromatography [Separation mechanism] Difference in hydrophobicity [Main applications] Purification of proteins *Unlike reversed-phase, proteins are not likely to become denatured. CSMSIL Protein-R CSMSIL HIC Separate based on differences in hydrophobicity Little loss in enzyme activity and the tertiary structure of proteins IV. Related Products Excellent separation High recovery rate utstanding stability at low ph Speci cations Packing Material Silica Gel Average Particle Size Average Pore Size Protein-R High purity porous spherical slica 5 m approx. 300 Speci c Surface Area approx. 150 m 2 /g Bonded Phase Bonding Type Main Interaction End-Capping Treatment ctadecyl group Polymeric Hydrophobic interaction ear-perfect treatment ph Range * Features High recovery rate Acid-resistant *ptimal ph range of silica-based columns is between 2 and 7.5. Extreme ph may signi cantly decrease column lifetime. 40

43 Comparison of Separation Protein-R shows sharper peaks for proteins than conventional C18 wide-pore columns. Comparison of Separation Property Column: Column size: 4.6mmI.D.-150mm Mobile phase: A; 0.05%TFA-20%Acetonitrile B; 0.05%TFA-60%Acetonitrile B conc % 20min Linear gradient Detection: UV220nm 1; Ribonuclease A (1.0 g) 2; Cytochrome C (1.0 g) 3; Lysozyme (1.0 g) 4; Albumin, Bovine [BSA] (1.0 g) 5; Myoglobin (1.0 g) 6; Albumin, Egg [valbumin] (1.0 g) rdering Information Analytical / Preparative Columns (Particle Size: 5 m) CSMSIL Protein-R Packed Column 2.0 x x x x CSMSIL Protein-R Guard Column CSMSIL C18-AR-300, C8-AR-300, C4-AR-300, Ph-AR-300 Wide-pore reversed-phase column 4 types of phases (octadecyl, octyl, butyl and phenyl) Speci cations Protein-R 10 x x x x C x x x Packing Material 5C18-AR-300 5C8-AR-300 5C4-AR-300 5Ph-AR-300 Silica Gel High purity porous spherical silica Aerage Particle Size 5 μm Average Pore Size 300 Å Speci c Surface Area 150 m 2 /g I. HPLC Columns II. SFC Columns III. Preparative Packing Materials IV. Related Products Bonded Phase Structure Si H Si H Si H Si H Bonded Phase ctadecyl group ctyl group Butyl group Phenyl group Bonding Type Polymeric Main Interaction Hydrophobic interaction Hydrophobic interaction - interaction End-Capping Treatment ear-perfect treatment ph Range * Carbon Content approx. 12% approx. 7% approx. 6% approx. 7% *ptimal ph range of silica-based columns is between 2 and 7.5. Extreme ph may signi cantly decrease column lifetime. 41

44 Comparison of Separation CSMSIL AR-300 packed column series offers 3 types of alkyl phases and a phenyl phase. I. HPLC Columns Comparison of Separation Property Column: Column size: 4.6mmI.D.-150mm Mobile phase: A; 0.05%TFA-20%Acetonitrile B; 0.05%TFA-60%Acetonitrile B conc % 20min Linear gradient Detection: UV220nm 5C 18 -AR-300 5C 8 -AR-300 5C 4 -AR-300 5Ph-AR-300 Hemoglobin, Bovine (10 g) II. SFC Columns III. Preparative Packing Materials IV. Related Products rdering Information Analyti al / Preparative Columns (Particle Size: 5 m) CSMSIL 5C18-AR-300 Packed Column 4.6 x x x CSMSIL 5C8-AR-300 Packed Column 4.6 x x x x x x x x x x x CSMSIL 5C18-AR-300 Guard Column 4.6 x x CSMSIL 5C8-AR-300 Guard Column 4.6 x x CSMSIL 5C4-AR-300 Packed Column CSMSIL 5C4-AR-300 Guard Column 4.6 x x x x x x x x x CSMSIL 5Ph-AR-300 Packed Column 4.6 x x x x x x x CSMSIL 5Ph-AR-300 Guard Column 4.6 x x

45 Gel Filtration Columns (Aqueous) CSMSIL Diol-120-II, Diol-300-II, Diol-1000-II Ideal for the size-based separation of proteins and water-soluble polymers Reduce undesirable adsorption Speci cations Packing Material 5Diol-120-II 5Diol-300-II 5Diol-1000-II Silica Gel High purity porous spherical silica (1) Average Particle Size 5 m Average Pore Size approx. 120 approx. 300 approx Å (2) Bonded Phase Diol group Target Substances Proteins, water soluble polymers Flow Rate (ml/min) Selection of Pore Size (protein) MW 5, ,000 MW 10, ,000 Selection of Pore Size (water-soluble polymers) MW 1,000-20,000 MW 5, ,000 MW 50, ,000 (1) With the silica-based gel, organic solvents, including methanol and acetonitrile, can be used. (2) If you require pore sizes greater than 1000 Å, please contact us. Calibration Curve Calibration Curve of Proteins Log MW Diol-300- Diol Elution Volume (ml) Linear pullulan calibration curve Log MW Diol-1000-II 5Diol-300-II 5Diol-120-II Elution Volume (ml) Column Mobile Phase Flow Rate Temperature CSMSIL 5Diol-II (7.5 mm l.d. x 600 mm) 20mmol/l Phosphate Buffer (ph7.0) 100mmol/l a2s04 1.0ml/min 30 o C Sample M.W. Sample M.W. Thyroglobulin 660,000 Peroxidase 40,000 Catalase 250,000 Carbonic Anhydrase 30,000 Glucose xidase 160,000 -Chymotrypsinogen A 25,700 Uricase 128,000 -Chymotrypsin 25,200 Choline xidase 95,000 Trypsinogen 24,000 Transferrin 85,000 Trypsin (bovine) 23,300 Conalbumin 77,500 Myoglobin 17,000 Malate Dehydrogenase 70,000 Lysozyme 14,300 -Glucosidase 68,500 Ribonuclease A 13,700 Albumin (BSA) 66,000 Cytochrome C 12,400 -Amylase 52,500 Aprotinin 6,500 Fetuin 48,000 Gly-Gly 132 Albumin (valbumin) 45,000 Column Mobile Phase Flow Rate Temperature Detection Sample Sample M.W. 1; P ,000 2; P ,000 3; P ,000 4; P ,000 5; P-50 48,000 6; P-20 23,700 7; P-10 12,200 8; P-5 5,800 9; Maltotriose ; Maltose ; Glucose 180 CSMSIL 5Diol- 7.5 mm I.D. x 300 mm) Water 1.0ml/min 30 o C RI Linear pullulan I. HPLC Columns II. SFC Columns III. Preparative Packing Materials IV. Related Products 43

46 Molecular Weight Range I. HPLC Columns Globular molecules have smaller apparent size compared to linear molecules of the same weight. Therefore, globular molecules can be separated with smaller pores compared to linear molecules. Fractionation Range (MW) Globular protein 5Diol-120-II 5Diol-300-II 5, ,000 10, ,000 Linear water-soluble polymer 5Diol-120-II 5Diol-300-II 1,000-20,000 5, ,000 5Diol-1000-II 50, ,000 II. SFC Columns Application DA ,000 10, ,000 Molecular Weight 10,000,000 CSMSIL Application Data 5Diol-120-II 5Diol-300-II 5Diol-1000-II III. Preparative Packing Materials Column: 5Diol-***-II Column size: 7.5mmI.D.-300mm Mobile phase: 20mmol/l Phosphate buffer(ph7.0), 100mmol/l a 2 S 4 Temperature: 30 C Detection: UV260nm 1; dsda, 1000bp 2; dsda, 100bp 3; ssda, 20mer 4; Uracil rdering Information IV. Related Products Analytical / Preparative Columns (Particle Size: 5 m) CSMSIL 5Diol-120-II Packed Column 7.5 x x CSMSIL 5Diol-120-II Guard Column 7.5 x CSMSIL 5Diol-300-II Packed Column 7.5 x x CSMSIL 5Diol-300-II Guard Column 7.5 x CSMSIL 5Diol-1000-II Packed Column 7.5 x CSMSIL 5Diol-1000-II Guard Column 7.5 x

47 Ion Exchange Columns CSMGEL IEX Series Available in 3 different ion-exchange modes (Anion-exchange type, Cation-exchange type, Amphoteric ion-exchange type) Available for 3 different application areas (for Puri cation, for Ultra-fast analysis, for Precise analysis) For separation of biopolymers such as proteins or nucleic acids Speci cations Packing Material Type Q Type Q- Type S Type S- Type M Type M- Gel Hydrophilic polymer Average Particle Size 5 m Average Pore Size 1000 on-porous 1000 on-porous 1000 on-porous Functional Group -CH3 + (CH3)3 -(CH2)3S3 - -CH3 + (CH3)3 + -(CH2)3S3 - Protein Binding mg mg mg mg mg(bsa)/ml 6-10 mg(bsa)/ml Capacity BSA/ml-resin Human IgG/ml-resin mg(igg)/ml 5-9 mg(igg)/ml 4.6 x x x x 50 I.D. x Lengh (mm) 4.6 x x x x 100 Column Material PEEK Connection Waters Type Type of Packing Material CSMGEL IEX Series are available in amphoteric ion-exchange type in which two kinds of packing materials are mixed, as well as in widely used anion-exchange type and cation-exchange type. Average Pore Size Type of Packing Material Target Sample Porous (1000 ) on-porous Anion-Exchange Type Acidic proteins / DA Type Q Type Q- Cation-Exchange Type Basic proteins Type S Type S- Amphoteric Ion-Exchange Type All proteins Type M Type M- Comprehensive isolation of proteins by amphoteric ion-exchange type (Type M) The amphoteric ion-exchange type enables the simultaneous separation of both acidic and basic proteins in one application. CSMSIL Application Data Column: CSMGEL IEX Column size: 4.6mmI.D.-50mm Mobile phase: A: 20mmol/l Phosphate buffer(ph6.8) B: 20mmol/l Phosphate buffer(ph6.8) + 0.5mol/l acl B conc % 30min Linear gradient Flow rate: 0.5 ml/min Detection: UV220nm Type Q Type S Type M I. HPLC Columns II. SFC Columns III. Preparative Packing Materials IV. Related Products 1; Ribonuclease A (1.5 g) 2; Cytochrome C (1.5 g) 3; Trypsin Inhibitor (6.0 g) 4; -Galactosidase (6.0 g) AP-1174 Type of Column CSMGEL IEX columns are available for 3 types of applications: Application Pore Size Column For Puri cation Porous (1000 ) Type Q Type S Type M For Precise Analysis on-porous Type Q- Type S- Type M- For Ultra-Fast Analysis on-porous Type Q- Type S- 45

48 Porous packing materials have higher binding capacity for proteins than the non-porous type, which means that peak shape does not spread even with injection of a large volume of sample. Therefore they are highly suitable for puri cation of large samples. I. HPLC Columns CSMSIL Application Data Column: CSMGEL IEX Type S Column size: 4.6mmI.D.-50mm Mobile phase: A: 20mmol/l Phosphate buffer(ph6.8) B: 20mmol/l Phosphate buffer(ph6.8) + 0.5mol/l acl B conc % 30min Linear gradient Flow rate: 0.5 ml/min Detection: UV280nm Each protein 500 g 1; Ribonuclease A 2; Cytochrome C 3; Lysozyme 100 g II. SFC Columns III. Preparative Packing Materials 10 g AP-1185 on-porous packing materials reduce spreading of samples in packing materials, resulting in high resolution separation for precise analysis, such as quality control of antibody preparations. The longer column length also contributes to the sharper peaks. CSMSIL Application Data Column: CSMGEL IEX Column size: Mobile phase: A: 20mmol/l Phosphate buffer(ph6.8) B: 20mmol/l Phosphate buffer(ph6.8) + 0.5mol/l acl B conc % 30min Linear gradient Flow rate: 0.5 ml/min Detection: UV220nm 1; Ribonuclease A (1.65 g) 2; Cytochrome C (1.65 g) 3; Lysozyme (1.65 g) Type S (4.6mmI.D.-50mm) Type S- (4.6mmI.D.-100mm) AP-1181 on-porous packing materials are not much affected by high ow rate and thus the materials are suitable for fast analysis. The shorter column length contributes to the fast analysis. IV. Related Products CSMSIL Application Data Column: CSMGEL IEX Column size: Mobile phase: A: 20mmol/l Phosphate buffer(ph6.8) B: 20mmol/l Phosphate buffer(ph6.8) + 0.5mol/l acl B conc % Linear gradient Type S: 30min Type S-: 10min Flow rate: Type S: 0.5 ml/min Type S-: 1.0 ml/min Detection: UV220nm Type S (4.6mmI.D.-50mm) Type S- (4.6mmI.D.-30mm) 1; Ribonuclease A (1.65 g) 2; Cytochrome C (1.65 g) 3; Lysozyme (1.65 g) AP-1183 rdering Information 46 Ion Exchange Mode Product ame Application Anion-exchange Type Cation-exchange Type Amphoteric Ion-exchange Type CSMGEL IEX Type Q For Puri cation 4.6 x CSMGEL IEX Type Q- For Ultra-fast Analysis 4.6 x CSMGEL IEX Type Q- For Precise Analysis 4.6 x CSMGEL IEX Type S For Puri cation 4.6 x CSMGEL IEX Type S- For Ultra-fast Analysis 4.6 x CSMGEL IEX Type S- For Precise Analysis 4.6 x CSMGEL IEX Type M For Puri cation 4.6 x CSMGEL IEX Type M-z For Precise Analysis 4.6 x

49 Hydrophobic Interaction Columns CSMSIL HIC Separate based on differences in hydrophobicity Little loss in enzyme activity and the tertiary structure of proteins Speci cations Packing Material HIC Silica Gel High purity porous spherical silica Average Particle Size 5 μm Average Pore Size approx. 300 Speci c Surface Area approx. 150 m 2 /g Main Interaction Hydrophobic interaction Applications A buffer with high salt concentration, usually 1-2 mol/l of (H4)2S4, is used as an initial mobile phase for adsorption of samples to a weakly hydrophobic stationary phase. The elution is done with a decreasing salt gradient. The application in the lower left shows that myoglobin elutes earlier than BSA under the buffer with high salt concentration, suggesting that myoglobin is less hydrophobic than BSA. Separation of Protein Standards CSMSIL Application Data Column: HIC Column size: 4.6mmI.D.-50mm Mobile phase: A:20mmol/l Phosphate Buffer +100mmol/l a 2 S mol/l (H 4 ) 2 S 4 (ph6.7) B:20mmol/l Phosphate Buffer +100mmol/l a 2 S 4 (ph6.7) B conc % 10min Linear gradient Detection: UV220nm 1; Myoglobin (1.0 g) 2; -Lactoglobulin (2.0 g) 3; Hemoglobin, Bovine (5.0 g) 4; Albumin, Bovine [BSA] (2.0 g) rdering Information Analytical Columns (Particle Size: 5 m) CSMSIL 5HIC Packed Column 4.6 x AP-0406 CSMSIL Application Data Column: HIC Column size: 4.6mmI.D.-50mm Mobile phase: A:20mmol/l Phosphate Buffer +100mmol/l a 2 S 4 +2mol/l (H 4 ) 2 S 4 (ph6.7) B:20mmol/l Phosphate Buffer +100mmol/l a 2 S 4 (ph6.7) B conc % 10min Linear gradient Detection: UV220nm -Glucosidase (15 g) AP-0407 I. HPLC Columns II. SFC Columns III. Preparative Packing Materials IV. Related Products 47

50 2 2 Si H3C CH3 (6) Columns for Fullerene Separation I. HPLC Columns Introduction Separation of fullerenes, especially preparative scale separation, on conventional HPLC columns is always problematic due to the low solubility and low recovery rate of fullerenes. CSMSIL offers a variety of columns designed for preparative scale separation of fullerenes, including higher fullerenes, metallofullerenes and fullerene derivatives. Speci cations Packing Material Buckyprep Buckyprep-D Buckyprep-M PBB PYE PE Silica Gel High purity porous spherical silica Average Particle Size 5 μm Average Pore Size approx. 120 Å Speci c Surface Area approx. 300 m 2 /g II. SFC Columns Bonded Phase Structure 2 2 S Br Br Br Br Br 2 III. Preparative Packing Materials Bonded Phase Pyrenylpropyl group itro-carbazoyl group Phenothiazinyl group Pentabromobenzyl group Pyrenylethyl group itrophenylethyl group Bonding Type Monomeric End-Capping Treatment ear-perfect treatment one ear-perfect treatment Carbon Content approx. 17% - approx. 13% approx. 8% approx. 18% approx. 9% Features H3C Comparison of Retention Si CH3 Standard column for fullerene separation. H3C Si CH3 For separation of derivatized fullerenes H3C Designed to separate metallofullerenes Designded for preparative separation of C60, C70 Separation of fullerene and structural isomers Separation of fullerene derivatives The figure below shows the retention time of C60 and C70 in toluene. CSMSIL fullerene separation columns (Buckyprep, Buckyprep-D, Buckyprep-M, PBB and PYE) exhibit high fullerene retention with toluene, so they can easily separate C60 and C70. Si CH3 H3C Si CH3 H3C Si CH3 H3C Si CH3 IV. Related Products Comparison of Retention Column: Column size: 4.6mmI.D.-250mm Mobile phase: Toluene Temperature: 30 C Detection: UV 325nm or 312nm C 60 C 70 Buckyprep 5PBB Buckyprep-M 5PYE Buckyprep-D 5C 18 -MS-II C 60 C Suggested Solvents for Fullerene Separation Solvent Solubility of C 60 (mg/ml) Features Toluene 3.2 The most commonly used solvent. n-hexane n-heptane -- Methanol Weaker eluent than toluene 2-Propanol -- Acetonitrile Weaker eluent than toluene. Recommended as a washing solvent for Buckyprep-D. Solvent Solubility of C 60 (mg/ml) Chlorobenzene 7.0 o-dichlorobenzene ,2,4- Trichlorobenzene 21.3 Features Stronger eluent than toluene. Recommended for higher fullerenes. Stronger eluent than chlorobenzene. Strongest eluent. Recommended as a washing solvent.

51 CSMSIL Buckyprep Standard column for fullerene separation Excellent separation for higher and derivatized fullerenes Difference in Preparative Separation Buckyprep can be used with toluene, the most commonly-used solvent in fullerene separation. Because tailing does not occur, you can inject about 35 times more sample than with a C18 column. Difference in Preparative Separation Column: Column size: 4.6mmI.D.-250mm Mobile phase: (Buckyprep) Toluene (C 18 -MS-II) Toluene/Acetonitrile = 55/45 Temperature: 30 C Detection: UV285nm Applications Fullerene toluene extract (2.5mg/ml) Higher Fullerenes CSMSIL Application Data Column: Buckyprep Column size: 4.6mmI.D.-250mm Mobile phase: Toluene Detection: UV312nm C 60 C 70 C 76 C 84 5C 18 -MS-II Buckyprep Inj.vol.: 4.5 l 70 l Inj.vol.: 4.5 l 2500 l AP-0412 xidized Fullerenes CSMSIL Application Data Column: Buckyprep Column size: 4.6mmI.D.-250mm Mobile phase: Toluene Detection: UV312nm C 60 C 60 AP-0413 I. HPLC Columns II. SFC Columns III. Preparative Packing Materials IV. Related Products rdering Information Analytical / Preparative Columns (Particle Size: 5 m) CSMSIL Buckyprep Packed Column CSMSIL Buckyprep Guard Column 4.6 x x x x x x x x

52 CSMSIL Buckyprep-D I. HPLC Columns For preparative separation of derivatized fullerenes For separation of derivatized fullerenes such as C60-indene (used for organic thin- lm solar cell) Applications Buckyprep-D offers improved separation for C60-indene, a derivatized fullerene that has received much attention as an n-type semiconductor material for organic thin- lm solar cells. CSMSIL Application Data Buckyprep Buckyprep-D II. SFC Columns Column: Column size: 4.6mmI.D.-250mm Mobile phase: Toluene Temperature: 30 C Detection: UV 325nm Inj.Vol.: C 60 [Indene] 2 (1.0mg/ml) 1.0μl Data courtesy of Yusuke Tajima, Dr. Sci. rganic ptoelectronics Laboratory, RIKE (Institute of Physics and Chemistry) III. Preparative Packing Materials Buckyprep-D retains derivatized fullerenes longer than C60. Therefore it is more suitable for preparative separation of derivatized fullerenes than our conventional Buckyprep column. CSMSIL Application Data Column: Column size: 4.6mmI.D.-250mm Mobile phase: Toluene Temperature: 30 C Detection: UV 325nm 1; C 60 (0.25mg/ml) 2; [6,6]-Phenyl-C 61 Butyric Acid Methyl Ester [PCBM] (0.25mg/ml) Inj.Vol. 1.0μl 5C 18 -MS-II Buckyprep Buckyprep-D IV. Related Products ote The baseline of Bukyprep-D is less stable relative to other fullerene columns. To stabilize baseline, let acetonitrile run through for 10 minutes before analysis. rdering Information Analytical / Preparative Columns (Particle Size: 5 m) CSMSIL Buckyprep-D Packed Column 4.6 x x x x CSMSIL Buckyprep-D Guard Column 4.6 x x x

53 CSMSIL Buckyprep-M Different selectivity from Buckyprep Excellent separation for metallofullerenes Applications Metallofullerenes CSMSIL Buckyprep-M is a phenothiazinyl-bonded silica-based column specifically designed for metallofullerene separation. Metallofullerenes are retained more strongly than other fullerenes on this column. CSMSIL Buckyprep-M is also effective for the separation of higher fullerenes and fullerene derivatives. CSMSIL Application Data Column: Column size: 4.6mmI.D.-250mm Mobile phase: Toluene Flow rate: 1.0 ml/min Detection: UV312nm Sc 76 (I) Sc 78 Sc 80 (I) C 86 Sample courtesy of Prof. H.Shinohara,Department of chemistry, agoya University CSMSIL Application Data Column: Column size: 4.6mmI.D.-250mm Mobile phase: Toluene Flow rate: 1.0 ml/min Detection: UV312nm Gd@C 82 (I) Sample courtesy of Prof. H.Shinohara,Department of chemistry, agoya University rdering Information Buckyprep Buckyprep Analytical / Preparative Columns (Particle Size: 5 m) CSMSIL Buckyprep-M Packed Column CSMSIL Buckyprep-M Guard Column 4.6 x x x x C86 Gd@C82(I) Sc2@C80-C76 C88, C x x x x Buckyprep-M C84 C86 Sc2@C76(I) Buckyprep-M Gd@C82(I) C88, C90 Sc2@C78 Sc2@C80(I) I. HPLC Columns II. SFC Columns III. Preparative Packing Materials IV. Related Products 51

54 CSMSIL PBB I. HPLC Columns Can be used with o-dichlorobenzene or carbon disul de Suitable for preparative scale separation Applications The loading capacity of CSMSIL PBB for C60 and C70 can be three times greater than CSMSIL Buckyprep. Preparative Separation of Fullerenes PBB Buckyprep II. SFC Columns Column: Column size: 20mmI.D.-250mm Mobile phase: Toluene Flow rate: 18 ml/min Temperature: Room temperature Detection: UV285nm Crude fullerenes (3.5mg/ml) III. Preparative Packing Materials IV. Related Products rdering Information Analytical / Preparative Columns (Particle Size: 5 m) CSMSIL 5PBB Packed Column 4.6 x x x CSMSIL PE Different selectivity from Buckyprep or PBB Excellent separation for derivatized fullerenes Applications CSMSIL 5PBB Guard Column 4.6 x x x PCBM, PCBB CSMSIL PE retains derivatized C60 stronger than C60. CSMSIL Application Data Buckyprep PBB PE Column: Column size: 4.6mmI.D.-250mm Mobile phase: (Buckyprep, PBB) Toluene (PE) Toluene/ Hexane = 25/75 Detection: UV325nm 1; [6,6]-Phenyl-C 61 Butyric Acid Methyl Ester [PCBM] (1.5 g) 2; [6,6]-Phenyl-C 61 Butyric Acid Butyl Ester [PCBB] (1.5 g) 3; C 60 (1.5 g) Hexane added to mobile phase due to PE's weak retention. 52

55 rdering Information Analytical / Preparative Columns (Particle Size: 5 m) CSMSIL 5PE Packed Column CSMSIL 5PE Guard Column 4.6 x x x x CSMSIL PYE rdering Information Analytical / Preparative Columns (Particle Size: 5 m) CSMSIL 5PYE Packed Column 4.6 x x x x Fullerene Chromatogram Index 4.6 x x x CSMSIL 5PYE Guard Column 4.6 x x x Fullerene Chromatogram Index includes more than 100 chromatograms. If you are interested in this index, please feel free to us at info.intl@nacalai.com. The online version is available at the website of The Fullerenes, anotubes and Graphene Research Society below. Fullerene Chromatogram Index C78 CAS o.: Molecular formula: C78 Column: Buckyprep Column size: 4.6 mmi.d.-250 mm Mobile phase: Toluene Flow rate: 1.0 ml/min Temperature: 30 Detection: UV 285 nm Attenuation: 0.64 aufs Sample conc.: 0.20 mg/ml Injection volume: 10.0 l The Fullerenes, anotubes and Graphene Research Society I. HPLC Columns II. SFC Columns III. Preparative Packing Materials IV. Related Products Data courtesy of Website: 53

56 I. HPLC Columns (7) Columns for Soluble Carbon anotube Separation CSMSIL CT-300, CT-1000, CT-2000 Size-based separation of soluble carbon nanotubes Three pore sizes (300, 1000, 2000 ) High durability Speci cations II. SFC Columns III. Preparative Packing Materials Packing Material CT-300 CT-1000 CT-2000 Silica Gel High purity porous spherical silica Average Particle Size 5 m Average Pore Size approx. 300 approx approx Bonded Phase Hydrophilic group (neutral) ph Range Pressure 15 MPa and below Applications Carbon anotubes CSMSIL CT columns offer improved separation for DA-wrapped carbon nanotubes by connecting three columns with different pore sizes. CSMSIL Application Data Column: CT CT CT-2000 Column size: 7.5mmI.D.-300mm 3 Mobile phase: 0.5mmol/l EDTA, 200mmol/l acl, 40mmol/l Tris-Tris HCl(pH7.0) Temperature: 40 C Detection: UV260nm( 1), 350nm( 20) Inj. Vol. DA-wrapped CT(CT-DA) 500 l Fr.18(17-18min) Fr.20(19-20min) Fr.22(21-22min) Fr.24(23-24min) Full Scale (2.5 m) IV. Related Products Data courtesy of Prof. H.Shinohara, Dr. Y.Asada, Department of chemistry, agoya University rdering Information Analytical Columns (Particle Size: 5 m) CSMSIL CT-300 Packed Column 7.5 x CSMSIL CT-300 Guard Column 7.5 x AP-1147 CSMSIL CT-1000 Packed Column 7.5 x CSMSIL CT-2000 Packed Column 7.5 x CSMSIL CT-1000 Guard Column 7.5 x CSMSIL CT-2000 Guard Column 7.5 x

57 II. SFC Columns Supercritical Fluid Chromatography (SFC) has become more attractive because it offers some advantages over HPLC, such as high speed, unique selectivity and environmentally friendly separations. Many conventional normal-phase stationary phases, such as diol, amino and cyano, have been used for SFC applications. However, these phases present limitations for separations. CSMSIL SFC Columns have been developed to enhance the capability of SFC separations. CSMSIL SFC Columns acalai Tesque has developed columns specially designed for SFC in collaboration with acalai USA and P zer, Inc. Global R&D: CSMSIL HP, PY (equivalent to 2-ethylpyridine) and Quinoline. In addition to these, our HPLC columns Cholester and PBr have been tested for use with SFC. Packing Material HP PY Quinoline Cholester PBr Average Particle Sizes 3, 5 μm 2.5 μm 5 μm 2.5 μm 5 μm 5 μm Average Pore Sizes Speci c Surface Area 300 m 2 /g 330 m 2 /g 300 m 2 /g 330 m 2 /g 300 m 2 /g 300 m 2 /g Bonded Phase Structure Bonded Phase H 3-Hydroxyphenyl group Pharmaceutical Analysis Each phase has different retention properties. Imipramine 200 CSMSIL 100 3HP 0 Si H Column: CSMSIL ** Column size: 2.1mmI.D.-150mm Mobile phase: A: C2 B: 0.1% CH3CH4 -Methanol B conc. 0 60% (0 14min), 60% (14-17min) Flow rate: 0.8 ml/min BPR: 10 MPa Temperature: 40 C Detection: UV260nm Imipramine (1mmol/l) Inj.Vol.: 2.0 μl H Si Pyridinyl group Quinoline group Cholesteryl group min Procaine CSMSIL 3HP 250 H3C H3C H3C Si CH3 Column: CSMSIL ** Column size: 2.1mmI.D.-150mm Mobile phase: A: C2 B: 0.1% CH3CH4 - Methanol B conc. 0 60% (0 14min), 60% (14-17min) Flow rate: 0.8 ml/min BPR: 10 MPa Temperature: 40 C Detection: UV280nm Procaine (1mmol/l) Inj.Vol.: 2.0 μl Br Br H3C Si Br CH3 Br Br Pentabromobenzyl group min H 2 I. HPLC Columns II. SFC Columns III. Preparative Packing Materials IV. Related Products 100 CSMSIL 3PY min CSMSIL 3PY min Ethylpyridine min 2-Ethylpyridine min 100 CSMSIL 5PBr min Data courtesy of Kyushu University Medical Institute of Bioregulation Research Center for Transomics Medicine Division of Metabolomics SFC-214 CSMSIL 5PBr min Data courtesy of Kyushu University Medical Institute of Bioregulation Research Center for Transomics Medicine Division of Metabolomics SFC

58 Comparison of Retention Behavior I. HPLC Columns The following three stationary phases were evaluated for their retention of hydrophilic, hydrophobic, acidic and basic compounds. CSMSIL HP and PY elute hydrophobic compounds rst and retain hydrophilic compounds longer, whereas PBr elutes in the reverse order, exhibiting high retention for hydrophobic compounds. HP had the longest retention for basic compounds. Column: CSMSIL ** Column size: 2.1mmI.D.-150mm Mobile phase: A: C2 B: 0.1% CH3CH4-Methanol B conc. 0 50% (0 14min), 50% (14-17min) Flow rate: 0.8 ml/min BPR: 10 MPa Temperature: 40 C Detection: ESI-MS/MS, MRM Sample conc. 8 μmol/l Inj.Vol.: 2.0 μl CSMSIL 3PY o.1 o.2 o.3 o.4 H CSMSIL 3HP H 3C H H H H H H H Uridine 2; 3,5 - Dibenzyloxyacetophenone H 3C S H CSMSIL 5PBr H 3C + 3; 4- Benzyltrimethyl Ethylbenzenesulfonic Acid ammonium II. SFC Columns III. Preparative Packing Materials (o.1) Hydrophilic Compound (o.2) Hydrophobic Compound (o.3) Acidic Compound (o.4) Basic Compound CSMSIL HP (3-Hydroxyphenyl) Applications min min min min min min min min min min Data courtesy of Kyushu University Medical Institute of Bioregulation Research Center for Transomics Medicine Division of Metabolomics min min IV. Related Products mau min Column size: Mobile phase: Gradient: Flow rate: Temperature: Detection: 4.6 mm I.D. x 150 mm Carbon Dioxide : Methanol 95:5 to 65:35 over 6 minutes 5.0 ml/min 30 o C UV 230 nm 1. Ibuprofen 2. Fenoprofen 3. Flurbiprofen 4. Ketoprofen 5. Indoprofen Beta Blockers (Peak elution order reversal under identical conditions) CSMSIL HP (3-Hydroxyphenyl) ethylpyridine Atenolol EIC m/z 267 H 3 C H Pindolol EIC m/z 249 H H Pindolol EIC m/z Atenolol EIC m/z 267 H 3 C H H H 2 min min Column size: Mobile phase : Gradient: Flow rate: Temperature: Detection: 4.6 mm I.D. x 150 mm Carbon Dioxide : Methanol w/20mm ammonium formate 95:5 to 50:50 over 4 minutes 5.0 ml/min 30 o C APCI(+) 1. Pindolol 2. Atenolol 56

59 rdering Information Analytical / Preparative Columns (Particle Size: 5 m) CSMSIL 5HP (3-Hydroxyphenyl) Packed Columns 2.0 x x x x CSMSIL PY (Pyridinyl) Applications Hydrophilic organics DCDP-pyrimidine caffeine rdering Information HAT Uracil Analytical / Preparative Columns (Particle Size: 5 m) CSMSIL 5PY (Pyridinyl) Packed Columns 2.0 x x x x CSMSIL 5HP (3-Hydroxyphenyl) Guard Columns 10.0 x Sulfanilamide CSMSIL 5PY (Pyridinyl) Guard Columns 10.0 x (Particle Size: 3 m) CSMSIL 3HP (3-Hydroxyphenyl) Packed Columns 2.0 x x Phase: Pyridinyl Column size: 4.6 mm I.D. x 150 mm Flow rate: 5.6 ml/min Pressure 140 bar Temperature: Ambient Mobile phase : Methanol gradient 50% 0.1 min; return to (Particle Size: 3 m) CSMSIL 3PY (Pyridinyl) Packed Columns Colu mn Size I.D. x Le ngth (mm) 2.0 x x I. HPLC Columns II. SFC Columns III. Preparative Packing Materials IV. Related Products 57

60 CSMSIL Quinoline I. HPLC Columns II. SFC Columns The structural similarities between polar lipids, such as cholesterol and related analogs, have posed chromatographic and spectrometric challenges to analysts interested in quantifying these potential biomarkers. CSMSIL Quinoline has been developed to improve the separation of these structural isomers utilizing the - interactions and structural rigidity of the naphthylethyl phase and the hydrogen bonding of the pyridine phase. Applications Steroids orm Phase: Column size: Mobile phase: Gradient: Flow rate: Pressure: Temperature: Detection: Quinoline 4.6 mmi.d. x 150 mm Carbon Dioxide (A) : Methanol (B) 2% - 7% B in 4 minutes 4.5 ml/min 160 bar 30 o C APCI(+) Sample H H H F H H H H H 3 C H H H H H H 3 C H H 1: Dexamethasone-21-acetate 2: Cortisone 3: Hydrocortisone 4: Prednisolone H H H H 0 III. Preparative Packing Materials Caffeine analogs mau min Phase: Quinoline Column size: 4.6 mmi.d. x 150 mm Mobile phase: Carbon Dioxide:Methanol = 95:5 Flow rate: 4.5 ml/min Pressure: 160 bar Temperature: 30 o C Detection: UV 260 nm H 3 C H C H 3 H IV. Related Products rdering Information min 1: Caffeine 2: Theobromine 3: Etofylline Analytical / Preparative Columns (Particle Size: 5 m) CSMSIL Quinoline Packed Column 2.0 x 150 Inquire 4.6 x 100 Inquire 4.6 x 150 Inquire 10.0 x 150 Inquire 20.0 x 150 Inquire (Particle Size: 2.5 m) CSMSIL Quinoline Packed Column 3.0 x 50 Inquire 3.0 x 100 Inquire 3.0 x 150 Inquire 58

61 CSMSIL Cholester Fat-Soluble Vitamin Analysis When used with SFC, CSMSIL Cholester can separate fat-soluble vitamins and their impurities. Tocopheryl acetate (Vitamin E) CSMSIL Cholester exhibits strong retention for fat-soluble vitamins and is suitable for on-line SFE-SFC using Shimadzu s exera UC. The online extraction from food also produced triglyceride impurities, which were successfully separated from the vitamins. rdering Information For ordering information for CSMSIL Cholester, refer to page 24. CSMSIL PBr rdering Information Ergocalciferol (Vitamin D2) Retinyl palmitate (Vitamin A) For ordering information for CSMSIL PBr, refer to page 25. Ergocalciferol (Vitamin D2) Column: Column size: Mobile phase: Flow rate: Temperature: Detection: Sample conc: Retinyl palmitate (Vitamin A) Tocopheryl acetate (Vitamin E) Phytonadione (Vitamin K) CSMSIL 5Cholester 4.6 mm I.D. x 250 mm IPA in C2 3.0 ml/min 40oC UV-VIS 2.0 mg/ml Data courtesy of Shimadzu Corporation I. HPLC Columns II. SFC Columns III. Preparative Packing Materials IV. Related Products 59

62 III. Preparative Packing Materials I. HPLC Columns ormal and Reversed Phase Packing Materials Introduction pen column chromatography is an excellent and easy technique for large-scale preparation and puri cation at low cost. CSMSIL offers both normal and reversed phase packing materials based on totally porous spherical silica, which provides higher separation, less pressure and higher reproducibility than irregular silica. Speci cations II. SFC Columns III. Preparative Packing Materials Packing Material C18-P C18-PREP Silica Gel 60 (eutral) Silica Gel High purity porous spherical silica Average Particle Size 75, 140 m 50, 75, 140 m 75, 140 m Average Pore Size approx.120 approx. 60 Speci c Surface Area approx. 300 m 2 /g approx. 500 m 2 /g Bonded Phase ctadecyl group one Carbon Content approx. 19% 0% Residual Silanol Group Yes one Application Selection Guide (Reversed Phase) pen column chromatography / Flash column chromatography Reversed phase chromatography Type of Chromatography Concentration of rganic Solvent Suitable Packing Materials pen Column Chromatography Flash Column Chromatography High (70% or more) Low (70% or less) 75C 18-PREP 140C 18-PREP 75C 18-P 140C 18-P ormal phase chromatography page 62 page 61 IV. Related Products Medium Pressure Column Chromatography 50C 18-PREP page 62 60

63 CSMSIL C18-P A new Water-Wet C 18 packing material for reversed phase open column chromatography Usable under 100% aqueous eluents Characteristic The external surface of the C18-P gel is coated with hydrophilic group to increase wettability of the gel, and the octadecyl group is bonded within the pore of the gel. Conventional reversed phase C18 packing materials are restricted to about 30-50% water in the mobile phase. The CSMSIL C18-P is a new Water-Wet C18 packing material developed for reversed phase open column chromatography. The C18-P material can be used in 100% aqueous eluents. Figure 1. Structure of C18-P Applications Figure 2. Packing material in water Left: C18-P Separation of hydrophilic compounds in aqueous solution CSMSIL Application Data Column: 75C 18 -P Column size: 20mmI.D.-250mm Mobile phase: Methanol/ H 2 = 30/70 Flow rate: 0.2 ml/min Temperature: Room temperature Detection: UV254nm 1; Theobromine (100mg) 2; Theophylline (100mg Silica Gel Hydrophilic Group Hydrophobic Group Conventional Product C18-P provides good resolution Can be used with low concentration of organic solvent on open, flash column chromatography. Right: C18-PREP oat up Use with 70% or more organic solvent on open, ash column chromatography. In reversed phase chromatography, hydrophilic compounds such as theobromine and theophylline could be separated under low concentration of organic solvent. The figure shows they are clearly separated by reversed open column chromatography with 70% water. I. HPLC Columns II. SFC Columns III. Preparative Packing Materials IV. Related Products rdering Information CSMSIL C18-P Product ame Average Particle Size PKG Size g CSMSIL 75C18-P 75 m g kg g CSMSIL 140C18-P 140 m g kg 61

64 CSMSIL C18-PREP I. HPLC Columns Standard reversed phase packing material for open chromatography Endcapped 3 particle sizes (50, 75, 140 m) Particle Size, Flow Rate and Theoretical Plate umber Because reversed phase chromatography employs a mobile phase of high viscosity such as methanol and water, the ow rate is lower than that of normal phase chromatography, which uses mobile phase of low viscosity such as hexane and ethyl acetate C18-PREP 75C18-PREP 40μm C18-PREP 75C18-PREP 40μm II. SFC Columns Theoretical plate number Flow rate (ml/min) III. Preparative Packing Materials IV. Related Products 0 0 Performance Evaluation Flow rate (ml/min) Figure 1. Flow rate against theoretical plate number Column size: 20 mm I.D. x 300 mm CSMSIL Application Data Column: 50C18-PREP Column size: 4.6mmI.D.-250mm (Closed column) Mobile phase: Methanol/Water = 60 / 40 Temperature: 30 C Detection: UV 254 nm Flow rate: 2 ml/min (Methanol solution) 1; Uracil (0.15 mg/ml) 2; Acetophenone (0.2 mg/ml) 3; Benzene (20 mg/ml) 4; Toluene (40 mg/ml) Inj. Vol: 5 μl Methanol (%) Figure 2. Concentration of methanol against ow rate Column size: 10 mm I.D. x 180 mm bed height (gravitational liquid ow) CSMSIL Application Data Column: 75C18-PREP Column size: 4.6mmI.D.-250mm (Closed column) Mobile phase: Methanol/Water = 30 / 70 Temperature: 30 C Detection: UV 254 nm Flow rate: 1 ml/min (Methanol solution) 1; Uracil (0.04 mg/ml) 2; Caffeine (0.5 mg/ml) 3; Phenol (2 mg/ml) Inj. Vol: 20 μl rdering Information CSMSIL C18-PREP Product ame Average Particle Size PKG Size g CSMSIL 50C18-PREP 50 m g kg g CSMSIL 75C18-PREP 75 m g kg g CSMSIL 140C18-PREP 140 m g kg 62

65 Silica Gel (Spherical, eutral) The ph of silica gel is adjusted to neutral Suitable for the separation of ph-sensitive compounds Comparison with Conventional Silica Gel CSMSIL Application Data Column: Column size: 4.6mmI.D.-250mm Mobile phase: Hexane/Ethyl Acetate = 99/1 Detection: UV254nm 1; Methyl Benzoate(Standard) (10mg/ml) 2; Sample A (100mg/ml) Inj.Vol. 3 l rdering Information Silica gel 60 (Spherical, eutral) Product ame Average Particle Size PKG Size Silica Gel 60 (Spherical, eutral) for Column Chromatograph Sample A CSMSIL Application Data Column: Column size: 4.6mmI.D.-250mm Mobile phase: Hexane/Ethyl Acetate = 99/1 Detection: UV254nm 1; Methyl Benzoate(Standard) (10mg/ml) 2; Sample B (200mg/ml) Inj.Vol. 3 l Sample -- B 75 m 140 m ur conventional product ur conventional product g g kg kg kg g g kg Silica Gel 60 (spherical, neutral) Silica Gel 60 (spherical, neutral) I. HPLC Columns II. SFC Columns III. Preparative Packing Materials IV. Related Products 63

66 Silica Gel (for Column Chromatography) I. HPLC Columns rdering Information Silica Gel (Spherical) Product ame Average Particle Size Average Pore Size Grade PKG Size Silica Gel 60, Spherical approx. 70 ~ 230 mesh 60 SP kg kg Silica Gel 120, Spherical approx. 70 ~ 230 mesh 120 SP kg II. SFC Columns Silica Gel (Irregular) Product ame Average Particle Size Average Pore Size Grade PKG Size g approx. 70 ~ 230 mesh SP kg kg Silica Gel kg g approx. 230 ~ 400 mesh SP kg kg IV. Related Products III. Preparative Packing Materials 64

67 IV. Related Products 1. Reagents for Mobile Phase Preparation Phosphate Buffer Solution (ph 2.5) (5X) ph-adjusted Filtered (0.2 m) How to Prepare Dilute this product with HPLC grade distilled water (1 part buffer solution : 4 parts water) to make the 20 mmol/l phosphate buffer used in the following CSMSIL applications. CSMSIL Chromatogram Index Clindamycin CAS o.: [ ] Molecular formula: C 18 H 33 Cl 2 5 S Column: 5C 18 -MS-II Column size: 4.6mmI.D.-150mm Mobile phase: Methanol/ 20mmol/l Phosphate buffer(ph7.0)=70/30 Dt Detection: ti UV210 nm Attenuation: aufs Sample conc.: 1.0mg/ml Injection volume: 30 l 3.0 l Retention time: 9.86min Capacity factor: 4.99 rdering Information Phosphate Buffer Solution (5X) Product ame Grade PKG Size Phosphate Buffer Solution (ph 2.5) (5X) SP L Phosphate Buffer Solution (ph 7.0) (5X) SP L Stock Solutions for HPLC rdering Information UV uorescence tested Easily prepare the mobile phases used in CSMSIL applications Product ame Grade PKG Size 1mol/l-Ammonium Formate Solution SP ml 1mol/l-Ammonium Acetate Solution SP ml CSMSIL Chromatogram Index Cefaclor CAS o.: [ ] Molecular formula: C 15 H 14 Cl 3 4 S Column: 5C 18 -MS-II Column size: 4.6mmI.D.-150mm Mobile phase: Methanol/ 20mmol/l Phosphate buffer(ph2.5)=20/80 Dt Detection: ti UV220 nm Attenuation: aufs Sample conc.: 1.0mg/ml Injection volume: 05 l 0.5 l Retention time: 8.93min Capacity factor: 3.75 I. HPLC Columns II. SFC Columns III. Preparative Packing Materials IV. Related Products Premixed Eluents for HPLC rdering Information Product ame Grade PKG Size 0.1vol% Formic Acid-Acetonitrile SP L L 0.1vol% Formic Acid-Distilled Water SP L L 0.1vol% Tri uoroacetic Acid-Acetonitrile SP L L 0.1vol% Tri uoroacetic Acid-Distilled Water SP L 65

68 Additives I. HPLC Columns rdering Information Product ame Grade PKG Size Acetic Acid SP ml Formic Acid SP ml Phosphoric Acid, rtho SP ml x 1 ml Tri uoroacetic Acid SP x 1.5 ml x 3 ml ml II. SFC Columns III. Preparative Packing Materials IV. Related Products Ion-pair Reagents rdering Information For Basic Samples (R-S3 - a + ) Product ame R: Grade PKG Size Sodium 1-Butanesulfonate C4H9- SP g Sodium 1-Pentanesulfonate C5H11- SP g g Sodium 1-Hexanesulfonate C6H13- SP g g Sodium 1-Heptanesulfonate C7H15- SP g g Sodium 1-ctanesulfonate C8H17- SP g g Sodium 1-onanesulfonate C9H19- SP g Sodium 1-Decanesulfonate C10H21- SP g Sodium 1-Dodecanesulfonate C12H25- SP g Sodium Lauryl Sulfate ** SP g 0.5M Solution Sodium 1-Butanesulfonate C4H9- SP x 10 ml Sodium 1-Hexanesulfonate C6H13- SP ml x 10 ml Sodium 1-ctanesulfonate C8H17- SP ml x 10 ml For Acidic Samples (C4H9)4 + X - Product ame X - : Grade PKG Size Tetra-n-butylammonium Bromide -Br SP g Tetra-n-butylammonium Chloride -Cl EP g g Tetra-n-butylammonium Hydrogensulfate -HS4 GR g Tetra-n-butylammonium Iodide -I SP g g Tetra-n-butylammonium Perchlorate -Cl4 SP g g Tetra-n-butylammonium Phosphate -H2P4 SP g 0.5M Solution Tetra-n-butylammonium Phosphate -H2P4 SP ml x 10 ml 66

69 2. Products for Sample Preparation Cosmonice Filter For sample ltration ust attach a lter on top of a syringe W Series (aqueous solution) The W series uses a new material of low-adsorptive and low-extractive PVDF (poly vinylidenedifluoride) filter, which can be used with various solvents. They are able to minimize the loss of proteins in the small amount of sample, and prevent secondary contamination during pre ltration. S Series (organic solvents) The S series uses a PTFE (poly tetra uoroethylene) lter, which shows strong resistance for solvents, acids, and alkalis. It is best for pre ltration of samples extracted with solvents such as chloroform and tetrahydrofuran. rdering Information Cosmonice Filter Product ame Cosmonice Filter W (aqueous) Cosmonice Filter S (solvent) Diameter (mm) Pore Size ( m) Connection Inlet: luer-lock; utlet: luer-slip, Connectable to needles Housing : polyethylene Cosmospin Filter For sample ltration Easy to use by centrifugation mnipore hydrophilic PTFE membrane lter Process Volume Hold-up Volume Product umber PKG Size ml or less < 10 l pkg ~10 ml < 30 l pkg ml or less < 10 l pkg ~10 ml < 30 l pkg I. HPLC Columns II. SFC Columns III. Preparative Packing Materials IV. Related Products rdering Information Cosmospin Filter Product ame Cosmospin Filter G Cosmospin Filter H Pore Size ( m) Maximum Sample Volume Dimensions: Membrane: Sample reservoir and collection tube: Hold-up Volume Maximum Centrifugal Force Rotor Size ( xedangle) Filtration Area Color Product umber PKG Size ml 5 l 5000 x g 1.5 ml 0.2 cm 2 Brown pkg ml 5 l 5000 x g 1.5 ml 0.2 cm 2 White pkg 10.6 mm diameter x 45 mm mnipore hydrophilic PTFE Polypropylene 67

70 Labeling Reagents I. HPLC Columns rdering Information Product ame Grade Storage PKG Size Dabsyl Chloride SP Room temp g 3,5-Dinitrobenzoyl Chloride (DBC) SP Dark and Cool g BD Chloride SP Refrigerator g g o-phthalaldehyde (PA) SP Refrigerator g g IV. Related Products III. Preparative Packing Materials II. SFC Columns 68

71 3. Column Care Products Introduction It is important to preserve a column by washing it with suitable cleaning methods before storing it under appropriate conditions to obtain stable data and prolong the column lifetime. Applicable Columns Cleaning Solution Kit and Storage Solution for Reversed Phase HPLC Columns is only applicable to reversed phase HPLC columns, such as CSMSIL C18-MS-II, AR-II, PAQ, EB, Cholester, AP, PYE, PBr and CSMCRE 2.6C18, 2.6Cholester and 2.6PBr. Please note that this product is not suitable for Sugar-D, HILIC, normal phase or ion exchange columns. Cleaning Solution Kit for Reversed Phase HPLC Columns Components Product ame Main Components PKG Size Quantity Container Cleaning Solution A Methanol 500 ml 2 Brown Glass Bottle Cleaning Solution B Tetrahydrofuran, Methanol 500 ml 1 Brown Glass Bottle Application Cleaning Solution Kit for Reversed Phase HPLC Columns is designed for washing away contaminant adsorption and stationary phase shedding. If you experience the following symptoms, please try their corresponding solution rst. Trouble Cause Solution Increase of pressure The baseline is not stable Split peak Contamination of sample in a column Silica gel damage Contamination of sample in a column Detachment of stationary phase Contamination of sample in a column Deterioration of column Wash with the column cleaning kit Change to a new column Wash with the column cleaning kit Wash with the column cleaning kit Wash with the column cleaning kit Change to a new column I. HPLC Columns II. SFC Columns III. Preparative Packing Materials IV. Related Products Procedure (For 4.6 mm I.D. x 150 mm) 1 Replace solvent with HPLC-grade distilled water (1 ml/min, 30 min). (*This step is for mobile phases containing high concentration buffer. If you are using a salt-free mobile phase, please start from step (2).) 2 Run the Cleaning Solution A through the column for 15 min at a ow rate of 1ml/min. 3 Run the Cleaning Solution B through the column at a ow rate of 1ml/min until the baseline becomes stable (approx. 15 min). 4 Run the Cleaning Solution A through the column for 15 min. The column is ready for storage. 69

72 Example of pressure difference between washed and unwashed columns IV. Related Products III. Preparative Packing Materials II. SFC Columns I. HPLC Columns The gure shows a pressure comparison between washed and unwashed columns using Cleaning Solution Kit. Repeated analysis of natural products was conducted using CSMSIL 5C18-MS-II (4.6 mm I.D. x 150 mm). Pressure (MPa) Analysis Times (Condition) Column: CSMSIL 5C18-MS-II 4.6 mm I.D. x 150 mm) Mobile phase: Methanol / H2 = 70 / 30 Flow rate: 1.0ml/min Temperature: 40 o C As shown in the gure above, the column pressure increases if you use column continuously without washing. If you wash the column, you can extend the column life time and ease the pressure burden on your HPLC equipment. Example of a Stable Baseline The baseline may be unstable if sample components with very long retention remain in the column or stationary phase shedding occurs. Especially when analyzing crude samples that have components with a wide range of chemical characteristics, some unwanted components may be strongly retained in the column and slowly elute out in subsequent runs. The resulting unstable baseline can be eliminated by washing the column with the Cleaning Solution Kit. mabs 10 rdering Information Product ame Grade PKG Size Cleaning Solution for Reversed Phase HPLC Columns SP kit Storage Solution for Reversed Phase HPLC Columns Storage Solution for Reversed Phase HPLC Columns is designed for storing columns under suitable conditions. Procedure nm 1.00 Before Washing min without Washing with Washing mabs nm 1.00 After Washing min (For 4.6 mm I.D. x 150 mm) (1) Replace solvent with HPLC-grade distilled water. (1 ml/min, 30 min) (*This step is for mobile phases containing high concentration buffer. If you are using a salt-free mobile phase, please start from step(2).) (2) Run the Storage Solution through the column for 15 min at a ow rate of 1ml/min, and store. rdering Information Product ame Grade PKG Size Storage Solution for Reversed Phase HPLC Columns SP kit (500 ml) 70

73 4. CSMSIL HPLC Accessories rdering Information CSMSIL Guard Cartridge Holder I.D. PKG Size 2.0 mm PKG 4.6 mm PKG PKG Size PKG PKG Size PKG CSMSIL Column Connecting Tube I.D. PKG Size 0.1 mm PKG 0.25 mm PKG Guard Cartridge Holder is required for Guard Cartridge. CSMSIL Column Pre lter employs lter with smaller pore size (1 m) than that of column frit (2 m). Column spare lter for pre lter I. HPLC Columns II. SFC Columns III. Preparative Packing Materials IV. Related Products For connecting columns 71

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