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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. Uracil & Phenol. Phenol. Uracil. Biphenyl. Flow rate: 1ml/mn Column: 75 x 4.6mm id

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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 Uracil & Phenol Phenol Uracil Biphenyl Flow rate: 1ml/mn Column: 75 x 4.6mm id Isocratic: 60% - 0.04% v/v H3PO4 40%ACN Toluene Toluene Biphenyl Type B Silica TYPE-C Silica™

  4. Cogent UDC-Cholesterol on 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. Cogent UDC-Cholesterol on TYPE-C Silica™ Phenol & Pyridine Neutral Acid 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

  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. Solvents: A: 10% B: 90% Solvents: A: 8% B: 92% Cogent UDC-Cholesterol on TYPE-C Silica™ Normal Phase Resolution of Basic Antidepressants Solvent A: 20mM NH4 COOH/NH4OH Solvent B: Acetonitrile pH 8.00

  12. RETENTION TIME MAP UDC-CHOLESTEROL Showing Both Normal and Reverse Phase Utility Column Dimensions 150x4.6mm

  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 RUNS 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…TYPE- C Silica™-unbondedcompared toCogent-UDC Cholesterol™

  16. Aqueous Reverse Phase & Organic Normal Phase of Carvone on TYPE-C Silica™- Unbonded AQUEOUS Reverse Phase = MeOH AQUEOUS Reverse Phase = ACN 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 4.6mm id UV: 255nm A: 90% E: 10% A: 80% B: 20% 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 METFORMIN 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 RUNS 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 TYPE-C Silica™-unbonded 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 Type-C Silica™-unbonded 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 TYPE-C Silica™-unbonded 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 TYPE-C Silica™ - unbonded 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 TYPE-C Silica™-unbonded, 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 Method Conditions 80% 0.05% v/vH3PO4 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/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 After 3 hoursof 100% aqueous exposure, results are the same as with the first injection 1- OXALIC ACID 2- TARTARIC ACID 3- FORMIC ACID 4- ACETIC ACID 5- SODIUM AZIDE 6- URACIL 7- FUMARIC ACID 8- PROPIONIC ACID 7 2 1 4 8 6 5 100% 0.05% v/v H3PO4 Flow Rate: 1ml/mn Column: 75 x 4.6mm id UV: 255

  28. USING 100 % AQUEOUS ON pH STABLE COGENT BIDENTATE C18™ WITH TYPE-C SILICA™ 1 After 3 hoursof 100% aqueous exposure, results are the same as with the first injection 1- OXALIC ACID 2- FORMIC ACID 3- ACETIC ACID 4- SODIUM AZIDE 5- URACIL 6- FUMARIC ACID 7- PROPIONIC ACID 6 4 2 3 5 7 100% Water w/0.05% v/v H3PO4 Flow Rate:1ml/mn Column: 75 x 4.6mm id UV: 255

  29. WATER SOLUBLE VITAMINS ON COGENT BIDENTATE C18™ WITH TYPE-C SILICA ™ Method Conditions 0-4 min: 95% 0.1% v/v TFA in DW 5% Acetonitrile 4-10 min: 85% 0.1% v/v TFA in DW 15% Acetonitrile 10-15 min: Equilibrate start conditions Flow Rate: 1ml/min Column: 250 x 4.6mm id Cogent Bidentate C18 UV: 270nm Peaks VitaminLogP 1- Niacinamide B3 - 0.45 2- Thiamine Mono-Nitrate B1 - 3.43 3- Pyridoxine HCl B6 - 0.56 4- Folic Acid B11 - 2.81 5- Riboflavin B2 - 1.05

  30. WHAT ABOUT PEPTIDES?

  31. PEPTIDE ANALYSIS ON COGENT UDC-CHOLESTEROL™ RT: 3.6 min 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 30%-Distilled Water 70% -Methanol RT: 2.4 min 20%-Distilled Water 70% -Methanol

  32. 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.

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

  34. THE ANSWER IS A SCOUT KIT ™

  35. Cogent Scout Kits™ 150mm x 4.6mm ID Scout Kit™ 75mm x 4.6mm ID Mini-Scout Kit™ 50mm x 3.0mm ID LC-MS Methods Kit™ 50mm 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™

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

  37. Why Use 5 Different Phases in Method Development?

  38. Cogent HPS  C18 (1) Phenol Isocratic Separation 1 2 + 4 (2) Dimethyl Phthalate 3mL/min 20mM NH4COOH & NH4OH pH 8.00 5 (3) Toluene 3 + 6 (4) Nortriptyline (5) Imipramine (6) Amitryptiline

  39. Cogent HPS Cyano (1) Phenol Isocratic Separation (2) Dimethyl Phthalate 3mL/min 20mM NH4COOH & NH4OH pH 8.00 1+2+3 (3) Toluene 4+5+6 (4) Nortriptyline (5) Imipramine (6) Amitryptiline

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

  41. Multi-Mode w/ Cogent HPSC18 and Cyano (1) Phenol Isocratic Separation (2) Dimethyl Phthalate 3mL/min 20mM NH4COOH & NH4OH pH 8.00 (1) (5) (4) (6) (3) Toluene (2) (4) Nortriptyline (3) (5) Imipramine (6) Amitryptiline

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

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

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