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 Type B Silica TYPE-C Silica™ Flow rate: 1ml/mn Column: 75 x 4.6 mm id Isocratic 60% % v/v H 3 PO 4 40%ACN

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

Cogent UDC-Cholesterol  on TYPE-C Silica™

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

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

Pyridine + Phenol + Neutral + Acidic pH 8.00 pH 2.18 pH % 20mM NH 4 COOH/NH 4 OH 60% (H 3 PO 4 ) 90% (H 3 PO 4 ) 95% Acetonitrile 40% Acetonitrile 10% Acetonitrile Peak Order UDC-Cholesterol  Column with TYPE-C Silica 

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

Cogent HPS  C18Cogent UDC-Cholesterol  Flow Rate : 3ml/min Wavelength : 254nm Solvents A: Buffer B: 100%ACN Basic Compounds

Basic Compounds Normal Phase Resolution of Basic Antidepressants Solvent B: Acetonitrile UDC-Cholesterol  Column with TYPE-C Silica  A: 10% B: 90% A: 8% B: 92% Solvent A: 20mM NH 4 COOH/NH 4 OH pH 8.00

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

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

COGENT UDC-CHOLESTEROL  w/ TYPE-C SILICA  A: 99.95% Aqueous % H 3 PO 4 (pH 2.18) B: 99.95% Acetonitrile % H 3 PO 4 Flow Rate: 1 ml/min Column: 75 x 4.6mm id UV : 210nm Back pressure: 600 psi Metformin- log P = Glyburide - log P = Same Column - ISOCRATIC By changing solvent composition, the elution order can be reversed I AQUEOUS 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?

Un-bonded Type-C Silica ™ Cogent-UDC Cholesterol ™ Let us review… Un-bonded Type-C Silica ™ compared to Cogent-UDC Cholesterol ™

Aqueous Reverse Phase & Organic Normal Phase of Carvone on Un-bonded Type-C Silica™ A: 80% B: 20% A: 90% C: 10% A: 90% D: 10% AQUEOUS Reverse Phase = THF A: 0.05%v/v H 3 PO 4 (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 E: 95% F: 5% ORGANIC Normal Phase=Hexane & Ethyl Acetate AQUEOUS Reverse Phase = MeOHAQUEOUS Reverse Phase = ACN

TYPE-C SILICA ™ CAN SEPARATE METFORMIN FROM GLYBURIDE METFORMIN GLYBURIDE 73% 0.05% v/v H 3 PO 4 27% Acetonitrile Flow:1ml/mn Column: 75 x 4.6mm id UV:255nm HOWEVER…

COGENT UDC-CHOLESTEROL  w/ TYPE-C SILICA  A: 99.95% Aqueous % H 3 PO 4 (pH 2.18) B: 99.95% Acetonitrile % H 3 PO 4 Flow Rate: 1 ml/min Column: 75 x 4.6mm id UV : 210nm Back pressure: 600 psi Metformin- log P = Glyburide - log P = Same Column - ISOCRATIC By changing solvent composition, the elution order can be reversed I AQUEOUS NORMAL PHASE: NON POLAR FIRST! II AQUEOUS REVERSE PHASE: POLAR FIRST!

MIXED SEPARATION MECHANISMS FOR DIFFERENT COMPOUNDS Aqueous Normal Phase Separations 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… Aqueous Reverse Phase separations of Glyburide. 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.

HOW DOES THIS WORK WITH METFORMIN? Aqueous Normal Phase Separations 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? 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 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.

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.

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.

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

SELECTIVITY COMPARISON IN AQUEOUS REVERSE PHASE OF URACIL, PYRINDINE, PHENOL Method Conditions 80% 0.05% v/v H 3 PO 4 20% Acetonitrile Flow:1ml/mn Column: 75 x 4.6mm id UV:255nm Method Conditions 90% 0.05% v/v H 3 PO 4 10% Acetonitrile Flow:1ml/mn Column: 75 x 4.6mm id UV:255nm ™ Cogent UDC-Cholesterol ™ bonded Type-C Silica™ un-bonded

SELECTIVITY COMPARISON IN AQUEOUS REVERSE PHASE OF AROMATIC ORGANIC ACIDS Method Conditions 80% 0.05% v/v H 3 PO 4 20% Acetonitrile Flow:1ml/mn Column: 75 x 4.6mm id UV:255nm Method Conditions 90% 0.05% v/v H 3 PO 4 10% Acetonitrile Flow:1ml/mn Column: 75 x 4.6mm id UV:255nm ™ Cogent UDC-Cholesterol ™ bonded Type-C Silica™ un-bonded

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

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

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 100% 0.05% v/v H 3 PO 4 Flow Rate:1ml/mn Column: 75 x 4.6mm id UV: After 3 hours of 100% aqueous exposure, results are the same as with the first injection

WHAT ABOUT PEPTIDES?

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

PEPTIDE ANALYSIS ON COGENT UDC-CHOLESTEROL™ 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. 30%- Distilled Water 70%- Methanol RT: 2.4 min. 10%- Distilled Water 90%- Methanol

SO WHICH TYPE-C™ PHASE SHOULD I CHOOSE?

THE ANSWER IS A SCOUT KIT ™

Cogent Scout Kits™ 150mm x 4.6mm IDScout Kit™ 75mm x 4.6mm IDMini-Scout Kit™ 50mm x 3.0mm ID 50 mm x 2.1mm IDLC-MS Methods Kit™ 20mm x 4.0mm IDBallistic LC-MS Kit™ 20mm x 2.0mm IDMini-bore Scout Kit™

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

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

Multi-Mode Chromatography Unlike Mixed Mode Chromatography Multi-Mode is reproducible…every time

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