CAN YOUR HPLC COLUMN DO THIS ?CAN YOUR HPLC COLUMN DO THIS ?

Type B SILICA TYPE-C SILICA Type B SILICA TYPE-C SILICA

A Reverse Phase Chromatography Comparison of A Reverse Phase Chromatography Comparison of Unbonded Type B and Type C SilicaUnbonded Type B and Type C Silica HPLC Columns HPLC Columns

Using an Aqueous Mobile Phase Using an Aqueous Mobile Phase

Type B SilicaType B Silica TYPE-C Silica™TYPE-C Silica™

Flow rate: 1ml/mn Column: 75 x 4.6 mm id

Isocratic 60% - 0.04% v/v H3PO4 40%ACN

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

Cogent UDC-CholesterolCogent UDC-Cholesterol on TYPE-C Silica™on TYPE-C Silica™

Cogent HPS C18 Cogent UDC-Cholesterol

Flow Rate: 3ml/min

Wavelength: 254nm

Solvents A: Buffer B: 100%ACN

Neutral CompoundsNeutral Compounds

Cogent HPS C18 Cogent UDC-Cholesterol

Flow Rate: 3ml/min

Wavelength: 254nm

Solvents A: Buffer B: 100%ACN

Acidic CompoundsAcidic Compounds

Pyridine + Phenol + Neutral + AcidicPyridine + Phenol + Neutral + Acidic

pH 8.00 pH 2.18 pH 2.185% 20mM NH4COOH/NH4OH 60% (H3PO4) 90% (H3PO4)95% Acetonitrile 40% Acetonitrile 10% Acetonitrile

Peak OrderPeak Order Peak Order

UDC-Cholesterol Column with TYPE-C Silica

Phenol-Pyridine CompoundsPhenol-Pyridine CompoundsCogent HPS C18 Cogent UDC-Cholesterol

Flow Rate: 3ml/min

Wavelength: 254nm

Solvents A: Buffer B: 100%ACN

Cogent HPS C18 Cogent UDC-Cholesterol

Flow Rate: 3ml/min

Wavelength: 254nm

Solvents A: Buffer B: 100%ACN

Basic CompoundsBasic Compounds

Basic CompoundsBasic CompoundsNormal Phase Resolution of Basic AntidepressantsNormal 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 NH4 COOH/NH4OH

pH 8.00

RETENTION TIME MAP URETENTION TIME MAP UDC-CHOLESTEROLDC-CHOLESTEROL 150 x 4.6 mm ID

REAL LIFE UNIQUE APPLICATION:REAL LIFE UNIQUE APPLICATION:

METFORMIN GLYBURIDE

THE CHALLENGE:Separate: LogP of –2.64 & +4.79… Isocratically

COGENT UDC-CHOLESTEROLCOGENT UDC-CHOLESTEROL w/ TYPE-C SILICA w/ TYPE-C SILICAA: 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 = -2.64Glyburide - log P = +4.79Same 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?

Let us review…

Un-bonded Un-bonded Type-C SilicaType-C Silica™™ compared to

Cogent-Cogent-UDC CholesterolUDC Cholesterol™™

Aqueous Reverse Phase & Organic Normal Phase of Aqueous Reverse Phase & Organic Normal Phase of Carvone on Un-bonded Type-C Silica™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 H3PO4(pH 2.15)

B: MeOHC: AcetonitrileD: THFE: HexaneF: Ethyl Acetate

Flow Rate:1ml/minColumn: 75 x 4.6 mm idUV: 255nm

E: 95%F: 5%

ORGANIC Normal Phase=Hexane & Ethyl Acetate

AQUEOUS Reverse Phase = MeOH AQUEOUS Reverse Phase = ACN

TYPE-C SILICA TYPE-C SILICA ™™ CAN SEPARATE CAN SEPARATE METFORMIN FROM GLYBURIDEMETFORMIN FROM GLYBURIDE

METFORMIN

GLYBURIDE

73% 0.05% v/v H3PO4

27% Acetonitrile

Flow: 1ml/mnColumn: 75 x 4.6mm idUV: 255nm

HOWEVER…HOWEVER…

COGENT UDC-CHOLESTEROLCOGENT UDC-CHOLESTEROL w/ TYPE-C w/ TYPE-C SILICASILICA

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 = -2.64Glyburide - log P = +4.79Same 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 MIXED SEPARATION MECHANISMS FOR DIFFERENT COMPOUNDSCOMPOUNDS

The Aqueous Normal Phase SeparationsAqueous Normal Phase Separations of Metformin on both bonded phaseCogent 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.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?HOW DOES THIS WORK WITH METFORMIN?

The Aqueous Normal Phase SeparationsAqueous 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?HOW DOES THIS WORK WITH GLYBURIDE?

The Aqueous Reverse Phase separation of Glyburide on the Un-bonded Aqueous Reverse Phase separation of Glyburide on the Un-bonded Type-C Silica™ column shows retention at 27% acetonitrile, but no retention Type-C Silica™ column shows retention at 27% acetonitrile, but no retention at 35%. With the bonded UDC-Cholesterol™ column, even at 80% at 35%. With the bonded UDC-Cholesterol™ column, even at 80% acetonitrile, the Glyburide is retained.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™ COGENT UDC-CHOLESTEROL™ SEPARATION MECHANISM FOR SEPARATION MECHANISM FOR

GLYBURIDEGLYBURIDE

It can be seen that the silica hydride has no It can be seen that the silica hydride has no reverse phase retention above 35% acetonitrile. reverse phase retention above 35% acetonitrile.

The reverse phase retention on the The reverse phase retention on the Cogent UDC-Cholesterol™ column above 50% Cogent UDC-Cholesterol™ column above 50% acetonitrile must, therefore, be totally a function of acetonitrile must, therefore, be totally a function of the bonded phase. the bonded phase.

This suggests that the retention characteristics of This suggests that the retention characteristics of the bonded phase must be taken into account the bonded phase must be taken into account when considering the retention by reverse phase, when considering the retention by reverse phase, as well as, the compound itself. as well as, the compound itself.

KEY DIFFERENCES OF KEY DIFFERENCES OF TYPE-C™ PHASESTYPE-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 SELECTIVITY COMPARISON IN AQUEOUS REVERSE PHASE OF NEUTRALS ON BONDED AND UN-BONDED PHASE OF NEUTRALS ON BONDED AND UN-BONDED

PHASESPHASES

Method Conditions60% 0.05% v/v H3PO4

40% AcetonitrileFlow: 1ml/mnColumn: 75 x 4.6mm idUV: 255nm

1 2 3 4

4321

Cogent UDC-Cholesterol ™™bonded

Type-C Silica™un-bonded

Method Conditions40% 0.05% v/v H3PO4

60% AcetonitrileFlow: 1ml/mnColumn: 75 x 4.6mm idUV: 255nm

SELECTIVITY COMPARISON IN AQUEOUS REVERSE SELECTIVITY COMPARISON IN AQUEOUS REVERSE

PHASE OF URACIL, PYRINDINE, PHENOLPHASE OF URACIL, PYRINDINE, PHENOL

Method Conditions80% 0.05% v/v H3PO4

20% AcetonitrileFlow: 1ml/mnColumn: 75 x 4.6mm idUV: 255nm

Method Conditions90% 0.05% v/v H3PO4

10% AcetonitrileFlow: 1ml/mnColumn: 75 x 4.6mm idUV: 255nm

1

1

1

2

2

3

3

Cogent UDC-Cholesterol™™bonded

Type-C Silica™un-bonded

SELECTIVITY COMPARISON IN AQUEOUS SELECTIVITY COMPARISON IN AQUEOUS

REVERSE PHASE OF AROMATIC ORGANIC ACIDSREVERSE PHASE OF AROMATIC ORGANIC ACIDS

Method Conditions80% 0.05% v/v H3PO4

20% AcetonitrileFlow: 1ml/mnColumn: 75 x 4.6mm idUV: 255nm

Method Conditions90% 0.05% v/v H3PO4

10% AcetonitrileFlow: 1ml/mnColumn: 75 x 4.6mm idUV: 255nm

1

1

2

2

3

3

Cogent UDC-Cholesterol™™bonded

Type-C Silica™un-bonded

WHAT ABOUTWHAT ABOUT PHASE COLLAPSE PHASE COLLAPSE

WITH WITH 100% AQUEOUS ON 100% AQUEOUS ON

TYPE-C™ PRODUCTS?TYPE-C™ PRODUCTS?

USING 100 % AQUEOUS ON USING 100 % AQUEOUS ON COGENT UDC-CHOLESTEROL™ WITHCOGENT UDC-CHOLESTEROL™ WITH TYPE-C SILICA™TYPE-C SILICA™

100% 0.05% v/v H3PO4

Flow Rate: 1ml/mnColumn: 75 x 4.6mm idUV: 2155

1- OXALIC ACID2- TARTARIC ACID3- FORMIC ACID4- ACETIC ACID5- SODIUM AZIDE6- URACIL7- FUMARIC ACID8- PROPIONIC ACID1

2

3

4

56

7

8

After 3 hours of 100% aqueous exposure, results are the same as with the first injection

USING 100 % AQUEOUS ON pH STABLEUSING 100 % AQUEOUS ON pH STABLE COGENT BIDENTATE C18™ WITH TYPE-C SILICA™ COGENT BIDENTATE C18™ WITH TYPE-C SILICA™

1- OXALIC ACID2- FORMIC ACID3- ACETIC ACID4- SODIUM AZIDE5- URACIL6- FUMARIC ACID7- PROPIONIC ACID

100% 0.05% v/v H3PO4

Flow Rate:1ml/mnColumn: 75 x 4.6mm idUV: 2155

1

2 3

4

5

6

7

After 3 hours of 100% aqueous exposure, results are the same as with the first injection

WHAT ABOUTWHAT ABOUT PEPTIDES? PEPTIDES?

PEPTIDE ANALYSIS ON COGENT UDC-CHOLESTEROL™ PEPTIDE ANALYSIS ON COGENT UDC-CHOLESTEROL™

Peptide 611 Amino Acid ResiduesCentered around Aspargine

Flow Rate: 0.5ml/mnColumn: 150 x 4.6mm idUV: 210nm

Peptide 911 Amino Acid ResiduesCentered around Aspargine

RT: 3.6 min.

RT: 2.4 min.

30%- Distilled Water70%- Methanol

20%- Distilled Water70%- Methanol

PEPTIDE ANALYSIS ON COGENT UDC-CHOLESTEROL™PEPTIDE ANALYSIS ON COGENT UDC-CHOLESTEROL™

Peptide 119 Amino Acid ResiduesCentered around Histidine

Flow Rate: 0.5ml/mnColumn: 150 x 4.6mm idUV: 210nm

Peptide 119 Amino Acid ResiduesCentered around Histidine

RT: 3.7 min.30%- Distilled Water

70%- Methanol

RT: 2.4 min.10%- Distilled Water90%- Methanol

SO WHICH SO WHICH TYPE-C™ PHASE TYPE-C™ PHASE

SHOULD I CHOOSE?SHOULD I CHOOSE?

THE ANSWER IS A THE ANSWER IS A SCOUT KIT ™ SCOUT KIT ™

Cogent Scout Kits™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™

Cogent Scout Kits™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 ChromatographyMulti-Mode Chromatography

Cogent HPS C18

Cogent HPS Cyano

Coupler

Multi-Mode ChromatographyMulti-Mode Chromatography

• Unlike Mixed Mode Chromatography

• Multi-Mode is reproducible…every time

Cogent Scout Kits™ Provide:Cogent Scout Kits™ Provide:

SpeedSelectivity ChoicesVersatilityMulti-Mode™ OptionMultiple Column Size Choices for LCMS