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 4.6mm id Isocratic: 60% % v/v H 3 PO 4 40%ACN Uracil & Phenol Toluene Biphenyl Uracil Phenol Toluene Biphenyl

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

Phenol & Pyridine Neutral Acid Phenol & Pyridine Neutral Acid 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 Cogent UDC-Cholesterol  on 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

Normal Phase Resolution of Basic Antidepressants Solvent B: Acetonitrile Cogent UDC-Cholesterol  on TYPE-C Silica™ Solvent A: 20mM NH 4 COOH/NH 4 OH Solvents: A: 10% B: 90% Solvents: A: 8% B: 92% pH 8.00

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

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 RUNS 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?

TYPE- C Silica ™- unbonded Cogent-UDC Cholesterol ™ Let us review… TYPE- C Silica ™- unbonded compared to Cogent-UDC Cholesterol ™

Aqueous Reverse Phase & Organic Normal Phase of Carvone on TYPE-C Silica™- Unbonded 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 4.6mm id UV: 255nm ORGANIC Normal Phase = Hexane & Ethyl Acetate AQUEOUS Reverse Phase = MeOHAQUEOUS Reverse Phase = ACN A: 80% B: 20% A: 90% E: 10% A: 90% D: 10% E: 95% F: 5%

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

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 RUNS 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 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… 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 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.

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 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 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.

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 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.

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 Method Conditions 40% 0.05% v/v H 3 PO 4 60% 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 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% Water w/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

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 min: Equilibrate start conditions Flow Rate: 1ml/min Column: 250 x 4.6mm id Cogent Bidentate C18 UV: 270nm Peaks Vitamin LogP 1- Niacinamide B Thiamine Mono-Nitrate B Pyridoxine HCl B Folic Acid B Riboflavin B

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 30%-Distilled Water 70% -Methanol RT: 2.4 min 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 IDLC-MS Methods Kit™ 50mm 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

Why Use 5 Different Phases in Method Development?

Cogent HPS  C18 3mL/min 20mM NH 4 COOH & NH 4 OH pH 8.00 (4) Nortriptyline (6) Amitryptiline (5) Imipramine (3) Toluene (2) Dimethyl Phthalate (1) Phenol Isocratic Separation

Cogent HPS  Cyano 3mL/min 20mM NH 4 COOH & NH 4 OH pH 8.00 (4) Nortriptyline (6) Amitryptiline (5) Imipramine (3) Toluene ( 2) Dimethyl Phthalate (1) Phenol Isocratic Separation

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

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

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