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CAN YOUR HPLC COLUMN DO THIS ?

CAN YOUR HPLC COLUMN DO THIS ?. Type B SILICA TYPE-C SILICA . A Reverse Phase Chromatography Comparison of Unbonded Type B and Type C Silica HPLC Columns Using an Aqueous Mobile Phase. Flow rate: 1ml/mn Column: 75 x 4.6 mm id Isocratic

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CAN YOUR HPLC COLUMN DO THIS ?

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  1. CAN YOUR HPLC COLUMN DO THIS ?

  2. Type B SILICA TYPE-C SILICA 

  3. A Reverse Phase Chromatography Comparison of Unbonded Type B and Type C Silica HPLC Columns Using an Aqueous Mobile Phase Flow rate: 1ml/mn Column: 75 x 4.6 mm id Isocratic 60% - 0.04% v/v H3PO4 40%ACN Type B Silica TYPE-C Silica™

  4. COGENT UDC-CHOLESTEROL w/ TYPE-C SILICA 

  5. Cogent UDC-Cholesterol on TYPE-C Silica™

  6. Cogent HPS C18 Cogent UDC-Cholesterol Neutral Compounds Flow Rate: 3ml/min Wavelength: 254nm SolventsA: Buffer B: 100%ACN

  7. Cogent HPS C18 Cogent UDC-Cholesterol Acidic Compounds Flow Rate: 3ml/min Wavelength: 254nm SolventsA: Buffer B: 100%ACN

  8. Pyridine + Phenol + Neutral + Acidic Peak Order Peak Order Peak Order pH 8.00 pH 2.18 pH 2.18 5% 20mM NH4COOH/NH4OH 60% (H3PO4) 90% (H3PO4) 95% Acetonitrile 40% Acetonitrile 10% Acetonitrile UDC-Cholesterol Column with TYPE-C Silica

  9. Cogent HPS C18 Cogent UDC-Cholesterol Phenol-Pyridine Compounds Flow Rate: 3ml/min Wavelength: 254nm SolventsA: Buffer B: 100%ACN

  10. Cogent HPS C18 Cogent UDC-Cholesterol Basic Compounds Flow Rate: 3ml/min Wavelength: 254nm SolventsA: Buffer B: 100%ACN

  11. Basic CompoundsNormal Phase Resolution of Basic Antidepressants A: 10% B: 90% A: 8% B: 92% Solvent A: 20mM NH4 COOH/NH4OH pH 8.00 Solvent B: Acetonitrile UDC-Cholesterol Column with TYPE-C Silica

  12. RETENTION TIME MAP UDC-CHOLESTEROL 150 x 4.6 mm ID

  13. METFORMIN GLYBURIDE REAL LIFE UNIQUE APPLICATION: THE CHALLENGE: Separate: LogP of –2.64 &+4.79… Isocratically

  14. COGENT UDC-CHOLESTEROL w/ TYPE-C SILICA A: 99.95% Aqueous - 0.05% H3PO4(pH 2.18) B: 99.95% Acetonitrile - 0.05% H3PO4 Flow Rate: 1 ml/min Column: 75 x 4.6mm id UV : 210nm Back pressure: 600 psi Metformin - log P = -2.64 Glyburide - log P = +4.79 Same Column - ISOCRATIC By changing solvent composition, the elution order can be reversed IAQUEOUS NORMAL PHASE: NON POLAR FIRST! II AQUEOUS REVERSE PHASE: POLAR FIRST! If 2 min. analysis with baseline resolution is too slow…WHY NOT INCREASE FLOW RATE?

  15. Let us review…Un-bonded Type-C Silica™compared toCogent-UDC Cholesterol™

  16. Aqueous Reverse Phase & Organic Normal Phase of Carvone on Un-bonded Type-C Silica™ AQUEOUS Reverse Phase = MeOH AQUEOUS Reverse Phase = ACN A: 80% B: 20% A: 0.05%v/v H3PO4(pH 2.15) B: MeOH C: Acetonitrile D: THF E: Hexane F: Ethyl Acetate Flow Rate: 1ml/min Column: 75 x 4.6 mm id UV: 255nm A: 90% C: 10% AQUEOUS Reverse Phase = THF ORGANIC Normal Phase=Hexane & Ethyl Acetate A: 90% D: 10% E: 95% F: 5%

  17. TYPE-C SILICA ™ CAN SEPARATE METFORMIN FROM GLYBURIDE 73% 0.05% v/v H3PO4 27% Acetonitrile Flow: 1ml/mn Column: 75 x 4.6mm id UV: 255nm METFORMIN GLYBURIDE HOWEVER…

  18. COGENT UDC-CHOLESTEROL w/ TYPE-C SILICA A: 99.95% Aqueous - 0.05% H3PO4(pH 2.18) B: 99.95% Acetonitrile - 0.05% H3PO4 Flow Rate: 1 ml/min Column: 75 x 4.6mm id UV : 210nm Back pressure: 600 psi Metformin - log P = -2.64 Glyburide - log P = +4.79 Same Column - ISOCRATIC By changing solvent composition, the elution order can be reversed IAQUEOUS NORMAL PHASE: NON POLAR FIRST! II AQUEOUS REVERSE PHASE: POLAR FIRST!

  19. MIXED SEPARATION MECHANISMS FOR DIFFERENT COMPOUNDS The Aqueous Normal Phase Separations of Metformin on both bonded phase Cogent UDC-Cholesterol™ and Un-bonded TYPE-C Silica™ Columns are the same. This suggests that the silica surface of both columns provides the main separation mechanism for Metformin regardless of the bonded phase. BUT… Compare this data to the Aqueous Reverse Phase separations of Glyburide. The bonded Cogent UDC-Cholesterol™ column, under the same conditions, separates Glyburide much differently from that of the un-bonded Type-C Silica™ column. This suggests that the bonded phase of the UDC-Cholesterol™ column provided the separation mechanism for Glyburide but not Metformin, under these conditions.

  20. HOW DOES THIS WORK WITH METFORMIN? The Aqueous Normal Phase Separations of Metformin at approximately 50% acetonitrile results in a longer retention time as the acetonitrile percentage increases. This occurs at the same percentage for both the un-bonded and bonded phases. This suggests that the silica surface of both columns provides the main separation mechanism for Metformin regardless of the bonded phase. HOW DOES THIS WORK WITH GLYBURIDE? The Aqueous Reverse Phase separation of Glyburide on the Un-bonded Type-C Silica™ column shows retention at 27% acetonitrile, but no retention at 35%. With the bonded UDC-Cholesterol™ column, even at 80% acetonitrile, the Glyburide is retained. The bonded Cogent UDC- Cholesterol™ column, under the same conditions, separates Glyburide much differently from that of the Un-bonded Type-C Silica™ column. This suggests that the bonded phase of the UDC-Cholesterol™ column provided the separation mechanism for Glyburide but not Metformin under these conditions.

  21. COGENT UDC-CHOLESTEROL™ SEPARATION MECHANISM FOR GLYBURIDE It can be seen that the silica hydride has no reverse phase retention above 35% acetonitrile. The reverse phase retention on the Cogent UDC-Cholesterol™ column above 50% acetonitrile must, therefore, be totally a function of the bonded phase. This suggests that the retention characteristics of the bonded phase must be taken into account when considering the retention by reverse phase, as well as, the compound itself.

  22. KEY DIFFERENCES OF TYPE-C™ PHASES A key issue to understanding the uniqueness of Type-C™ phases is that it is possible to have different compounds (Metformin and Glyburide) use different separation mechanisms (ANP & ARP) in the same analysis on the same column. Within the mechanisms above, the choice of un-bonded TYPE-C Silica™, bonded Cogent UDC-Cholesterol™ or bonded Cogent Bidentate C18™, can each produce a reverse phase response; which can be due to the silica hydride surface or the bonded phase as dominant factors in the separation or combinations of the two.

  23. SELECTIVITY COMPARISON IN AQUEOUS REVERSE PHASE OF NEUTRALS ON BONDED AND UN-BONDED PHASES Cogent UDC-Cholesterol ™ bonded Method Conditions 40% 0.05% v/v H3PO4 60% Acetonitrile Flow: 1ml/mn Column: 75 x 4.6mm id UV: 255nm 1 2 3 4 Type-C Silica™ un-bonded Method Conditions 60% 0.05% v/v H3PO4 40% Acetonitrile Flow: 1ml/mn Column: 75 x 4.6mm id UV: 255nm 1 2 3 4

  24. SELECTIVITY COMPARISON IN AQUEOUS REVERSE PHASE OF URACIL, PYRINDINE, PHENOL Cogent UDC-Cholesterol™ bonded Method Conditions 80% 0.05% v/vH3PO4 20% Acetonitrile Flow: 1ml/mn Column: 75 x 4.6mm id UV: 255nm 1 1 2 3 Type-C Silica™ un-bonded Method Conditions 90% 0.05% v/vH3PO4 10% Acetonitrile Flow: 1ml/mn Column: 75 x 4.6mm id UV: 255nm 1 2 3

  25. SELECTIVITY COMPARISON IN AQUEOUS REVERSE PHASE OF AROMATIC ORGANIC ACIDS Method Conditions 80% 0.05% v/v H3PO4 20% Acetonitrile Flow: 1ml/mn Column: 75 x 4.6mm id UV: 255nm Cogent UDC-Cholesterol™ bonded 1 2 3 Type-C Silica™ un-bonded Method Conditions 90% 0.05% v/v H3PO4 10% Acetonitrile Flow: 1ml/mn Column: 75 x 4.6mm id UV: 255nm 1 2 3

  26. WHAT ABOUT PHASE COLLAPSE WITH 100% AQUEOUS ON TYPE-C™ PRODUCTS?

  27. USING 100 % AQUEOUS ON COGENT UDC-CHOLESTEROL™ WITHTYPE-C SILICA™ 3 1- OXALIC ACID 2- TARTARIC ACID 3- FORMIC ACID 4- ACETIC ACID 5- SODIUM AZIDE 6- URACIL 7- FUMARIC ACID 8- PROPIONIC ACID After 3 hoursof 100% aqueous exposure, results are the same as with the first injection 7 2 1 4 8 100% 0.05% v/v H3PO4 Flow Rate: 1ml/mn Column: 75 x 4.6mm id UV: 2155 6 5

  28. USING 100 % AQUEOUS ON pH STABLE COGENT BIDENTATE C18™ WITH TYPE-C SILICA™ 1- OXALIC ACID 2- FORMIC ACID 3- ACETIC ACID 4- SODIUM AZIDE 5- URACIL 6- FUMARIC ACID 7- PROPIONIC ACID 1 After 3 hoursof 100% aqueous exposure, results are the same as with the first injection 6 4 3 2 100% 0.05% v/v H3PO4 Flow Rate:1ml/mn Column: 75 x 4.6mm id UV: 2155 5 7

  29. WHAT ABOUT PEPTIDES?

  30. PEPTIDE ANALYSIS ON COGENT UDC-CHOLESTEROL™ RT: 3.6 min. 30%- Distilled Water 70%- Methanol Peptide 6 11 Amino Acid Residues Centered around Aspargine Flow Rate: 0.5ml/mn Column: 150 x 4.6mm id UV: 210nm Peptide 9 11 Amino Acid Residues Centered around Aspargine RT: 2.4 min. 20%- Distilled Water 70%- Methanol

  31. PEPTIDE ANALYSIS ON COGENT UDC-CHOLESTEROL™ 30%- Distilled Water 70%- Methanol Peptide 11 9 Amino Acid Residues Centered around Histidine Flow Rate: 0.5ml/mn Column: 150 x 4.6mm id UV: 210nm Peptide 11 9 Amino Acid Residues Centered around Histidine RT: 3.7 min. 10%- Distilled Water 90%- Methanol RT: 2.4 min.

  32. SO WHICH TYPE-C™ PHASE SHOULD I CHOOSE?

  33. THE ANSWER IS A SCOUT KIT ™

  34. Cogent Scout Kits™ 150mm x 4.6mm ID Scout Kit™ 75mm x 4.6mm ID Mini-Scout Kit™ 50mm x 3.0mm ID 50 mm x 2.1mm ID LC-MS Methods Kit™ 20mm x 4.0mm ID Ballistic LC-MS Kit™ 20mm x 2.0mm ID Mini-bore Scout Kit™

  35. Cogent Scout Kits™ • Each Mini-Scout Kit™ contains: 5 HPLC Columns 2 Super-Link End Fittings 1 Super-Link Column Coupler 1 Instruction Manual

  36. Multi-Mode Chromatography Cogent HPS  C18 Coupler Cogent HPS Cyano

  37. Multi-Mode Chromatography • Unlike Mixed Mode Chromatography • Multi-Mode is reproducible…every time

  38. Cogent Scout Kits™ Provide: • Speed • Selectivity Choices • Versatility • Multi-Mode™ Option • Multiple Column Size Choices for LCMS

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