HPLC Column for Structural Isomers COSMOSIL Cholester

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1 HPLC Column for Structural Isomers COSMOSIL ol bonded stationary phase Increased stereoselectivity Improved resolution for geometric isomers Silica Gel Average Particle Size Average Pore Size Stationary Phase Main Interaction High Purity Porous Spherical Silica 5 m approx. 120 yl Group Hydrophobic Interaction Stereoselectivity Carbon Content approx. 20% Si O CH3 yl Group COSMOSIL is a reversed phase HPLC column with yl bonded silica packing material, which provides equivalent hydrophobicity like that of traditional ODS columns. However, offers strong stereoselectivity for hydrophobic compounds to yield unique and reproducible separation patterns following the same analytical conditions used with other ODS columns. Improved Separation yl bonded groups are very rigid structures and offer improved separation of compounds that are difficult to analyze with Alkyl Group bonded materials. Stereoisomers like Vitamin D CH 2 CH3 1.Vitamin D2 100% Methanol UV 265 nm 1. Vitamin D 2 (0.3 μg) 2. Vitamin D 3 (0.1 μg) CH 2 2.Vitamin D3 CH3 Comparison of Hydrophobicity COSMOSIL provides the same hydrophobicity as ODS and C30 columns. Separation of Alkylbenzene (CH 2 )n-h n = 0-5 Methanol : Water = 80 : Benzene 2. Toluene 3. Ethylbenzene 4. Propylbenzene 5. Butylbenzene 6. Amylbenzene

2 Characteristics of Separation Planarity Selectivity shows greater planarity selectivity. (Planarity : Terphenyl < Triphenylene) 1.Terphenyl 90% Methanol 1. Terphenyl 2. Triphenylene (0.10 μg) (0.01 μg) 2.Triphenylene Allyl Halides (Relative Strengths of Nucleophiles : Methyl < Chlorine < Bromine) COSMOSIL is more selective for Allyl Halides. CH3 1.Toluene C l Br 60% Methanol 1. Toluene 2. Chlorobenzene 3. Bromobenzene 2.Chlorobenzene 3.Bromobenzene Positional Isomers like Chloroacetophenones Generally the isomers of disubstituted benzene are difficult to separate with C18 because the structure and polar characteristics are very similar. In these cases COSMOSIL works very well. m, m, Cl COCH3 Cl Cl 50% Methanol 1. Chloroacetophenone (0.30 μg) 2. Chloroacetophenone (0.15 μg) 3. Chloroacetophenone (0.05 μg) COCH3 CO Geometrical Isomers like Stilbenes COSMOSIL offers improved resolution of geometric isomers. 1.cis-Stilbene 70% Methanol 1. cis-stilbene 2. trans-stilbene (0.1 μg) (0.2 μg) 2.trans-Stilbene

3 Application Data Vitamin A acid CO 1.13-cis-Retinoic Acid CO Methanol : 20mmol/l Phosphate Buffer (ph2.5) = 90 : 10 UV 350 nm cis-Retinoic Acid 2. all-trans-retinoic Acid (0.04 μg) (0.04 μg) 2. all-trans-retinoic Acid Carotene alpha-carotene THF : Methanol = 20 : 80 UV 470 nm 1. α-carotene 2. β-carotene beta-carotene Terphenyl Methanol : Water = 90 : Terphenyl 2. Terphenyl 3. Terphenyl (0.15 μg) (0.05 μg) (0.075 μg) Chlorophenol Cl Cl Cl Methanol : Water = 50 : Chlorophenol 2. Chlorophenol 3. Chlorophenol (4.0 μg) Methoxyphenol O O O o, o, Methanol : Water = 30 : Methoxyphenol 2. Methoxyphenol 3. Methoxyphenol

4 Catechins 5C18-MS-II Injection Vol. A: Acetonitrile : 20mmol/l Phosphate Buffer (ph2.5) = 10 : 90 B: Acetonitrile : 20mmol/l Phosphate Buffer (ph2.5) = 30 : 70 B 0 100%/20min Linear Gradient UV 280 nm 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) 1 μ (0.80 mg/ml) (0.08 mg/ml) (0.80 mg/ml) (0.40 mg/ml) (0.40 mg/ml) (0.20 mg/ml) (0.40 mg/ml) (0.20 mg/ml) (0.20 mg/ml) Further Separation Positional Isomers like Tolunitriles For positional isomers (e.g. Tolunitriles) that can not be analyzed efficiently with, the COSMOSIL NPE or COSMOSIL PYE columns offer advanced separation., ; 40% Methanol πnap; 50% Methanol 5PYE; 60% Methanol OR 1. Tolunitrile 2. Tolunitrile 3. Tolunitrile (1.0 μg) πnap 5PYE CN CN CN Ordering Information Product Name Product Number COSMOSIL Packed Column 1.0 mm I.D. x 150 mm mm I.D. x 250 mm mm I.D. x 30 mm mm I.D. x 50 mm mm I.D. x 100 mm mm I.D. x 150 mm mm I.D. x 250 mm mm I.D. x 150 mm mm I.D. x 250 mm lots set Product Name Product Number COSMOSIL Packed Column COSMOSIL Guard Column 4.6 mm I.D. x 250 mm mm I.D. x 150 mm mm I.D. x 250 mm mm I.D. x 150 mm mm I.D. x 250 mm mm I.D. x 250 mm mm I.D. x 10 mm mm I.D. x 20 mm mm I.D. x 20 mm mm I.D. x 50 mm mm I.D. x 50 mm Other size may be available. Please enquire.. Nijo Karasuma, Nakagyku, Kyoto , JAPAN TEL : +81-(0) FAX : +81-(0) Website : info.intl@nacalai.com For research use only, not intended for diagnostic or drug use. 1104

5 COSMOSIL ol bonded stationary phase Usable under condition the same as C18 Increased stereoselectivity and improved resolution for geometric isomers For separation of natural compounds Hydrophobic Interaction Figure shows the comparison of hydrophobic interactions with competitive C18 columns. provides 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. Comparison of Hydrophobic Interaction Mobile phase: Methanol/H 2 O = 80/20 Flow rate: : C : 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 Molecular Shape Selectivity The stationary phase of has very rigid structures and can distinguish different molecular shapes. offers improved separation for structurally similar compounds (C18 and C30). As in the following example retains planar Triphenylene longer than stereoscopic Terphenyl. Comparison of Molecular Shape Selectivity Mobile phase: Methanol/H 2 O = 90/10 Flow rate: : C : UV254nm : 1; Terphenyl (0.1 g) 2; Triphenylene (0.01 g) 5C18-MS- 5C18-AR- A company C18-1 B company C18 E company C18 A company C18-2 F company C18 G company C * Particle size: 5 μm(except F company: 3 μm) α(t/o),90% Methanol Figure. Comparison of molecular sharp selectivity AP-1018 I. HPLC Columns II. UHPLC Columns III. Preparative IV. Related Products V. Applications VI. Technical Notes VII. Index Packing Materials Terphenyl(O) Triphenylene(T) AP

6 I. HPLC Columns Improvement in Separation COSMOSIL provides enhanced selectivity over traditional C18 columns and offers greater performance in separating isomers or other closely related compounds. COSMOSIL is ideal for method development and serves as an excellent alternative to traditional C18 cult to separate with C18 and C30, but they are well resolved by COSMOSIL. Comparison with Competitors' C18 and C30 columns II. UHPLC Columns 5C18-MS- A Company C18-1 B Company C18 E Company C18 III. Preparative Packing Materials A Company C18-2 F Company C18 Good!! G Company C30 IV. Related Products V. Applications * Particle Size: 5 μm (except F Company: 3 μm) : 50% Methanol, : Chloroacetophenone 18 column. Both columns show good separation. However, sample loading capacity for preparative separations can be affected by a slight difference in separation ability. COS- MOSIL can load 4 times of sample volume compared with C18 columns. Comparison of Preparative Separation 5C 18 -AR-II VI. Technical Notes Mobile phase: Methanol Flow rate: : C : UV254nm : 1; Bibenzyl 2; Anthracene 10 l Injection 40 l Injection Bibenzyl Antharacene VII. Index 22

7 Applications COSMOSIL Application Data Mobile phase: A: Acetonitrile/ 20mmol/l Phosphate buffer(ph2.5) = 10/90 B: Acetonitrile/20mmol/lPhosphatebuffer(pH25)= buffer(ph2.5) 30/70 B conc % 20min Linear gradient Flow rate: : C : 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 COSMOSIL Application Data Ordering Information Analytical / Preparative Column Mobile phase: A: Acetonitrile/ 20mmol/l Phosphate buffer(ph2.5) = 20/80 B: Acetonitrile/ 20mmol/l Phosphate buffer(ph2.5) = 70/30 B conc % 20min Linear gradient Flow rate: : C : UV280nm : 1; Fisetin (0.25 g) 2; Myricetin (0.20 g) 3; 7,8-Dihydroxyflavone (0.05 g) 4; Luteolin (0.20 g) 5; Quercetin (0.20 g) 6; 7-Hydroxyflavone (0.10 g) 10; Chrysin (0.05 g) 7; Baicalein (0.05 g) 11; 6-Methoxyflavone (0.05 g) 8; 6-Hydroxyflavone (0.05 g) 12; 3-Hydroxyflavone (0.25 g) 9; Flavone (0.05 g) 13; 5-Hydroxyflavone (0.05 g) AP-1032 COSMOSIL Packed Column I.D. x Length (mm) 1.0 x x x x x x x x x please refer to page 62. I.D. x Length (mm) 4.6 x x lots set* x x x x x x *For 4.6 x lots set, please refer to page 11. AP-1072 COSMOSIL Guard Column I.D. x Length (mm) 4.6 x x x x x I. HPLC Columns II. UHPLC Columns III. Preparative IV. Related Products V. Applications VI. Technical Notes VII. Index Packing Materials 23