CAN YOUR HPLC COLUMN DO THIS ?
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Transcript of CAN YOUR HPLC COLUMN DO THIS ?
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 Silica™Unbonded Type B and TYPE-C Silica™ HPLC Columns HPLC Columns
Using an Aqueous Mobile PhaseUsing an Aqueous Mobile Phase
Type B SilicaType B Silica TYPE-C Silica™TYPE-C Silica™
Flow rate: 1ml/mn Column: 75 x 4.6mm id
Isocratic: 60% - 0.04% v/v H3PO4 40%ACN
Uracil & Phenol
Toluene
Biphenyl
Uracil
Phenol
Toluene
Biphenyl
Cogent UDC-CholesterolCogent UDC-Cholesterol on TYPE-C Silica™on 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
Phenol & Pyridine Neutral AcidPhenol & Pyridine Neutral Acid
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
Cogent UDC-CholesterolCogent UDC-Cholesterol on TYPE-C Silica™on 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
Normal Phase Resolution of Basic AntidepressantsNormal Phase Resolution of Basic Antidepressants
Solvent B: Acetonitrile
Cogent UDC-CholesterolCogent UDC-Cholesterol on TYPE-C Silica™on TYPE-C Silica™
Solvent A: 20mM NH4 COOH/NH4OH
Solvents:A: 10%B: 90%
Solvents:A: 8%B: 92%
pH 8.00
RETENTION TIME MAP URETENTION TIME MAP UDC-DC-CHOLESTEROLCHOLESTEROL
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 & +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 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?
Let us review…
TYPE- C SilicaTYPE- C Silica™-™-unbondedunbonded
compared to
Cogent-Cogent-UDC CholesterolUDC Cholesterol™™
Aqueous Reverse Phase & Organic Normal Phase of Aqueous Reverse Phase & Organic Normal Phase of Carvone on TYPE-C Silica™- UnbondedCarvone on TYPE-C Silica™- Unbonded
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 4.6mm idUV: 255nm
ORGANIC Normal Phase = Hexane & Ethyl Acetate
AQUEOUS Reverse Phase = MeOH AQUEOUS Reverse Phase = ACN
A: 80%B: 20%
A: 90%E: 10%
A: 90%D: 10%
E: 95%F: 5%
TYPE-C SILICATYPE-C SILICA™™ CAN SEPARATE CAN SEPARATE METFORMIN FROM GLYBURIDEMETFORMIN FROM GLYBURIDE
METFORMIN
73% 0.05% v/v H3PO4
27% Acetonitrile
Flow: 1ml/mnColumn: 75 x 4.6mm idUV: 255nm
HOWEVER…HOWEVER…
Glyburide
Metformin
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 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 MIXED SEPARATION MECHANISMS FOR DIFFERENT COMPOUNDSCOMPOUNDS
The Aqueous Normal Phase SeparationsAqueous Normal Phase Separations of Metformin on both bonded phaseCogent 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.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?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 TYPE-C Silica™-Aqueous Reverse Phase separation of Glyburide on the TYPE-C Silica™-unbonded column shows retention at 27% acetonitrile, but no retention at unbonded column shows retention at 27% acetonitrile, but no retention at 35%. With the bonded UDC-Cholesterol™ column, even at 80% acetonitrile, 35%. With the bonded UDC-Cholesterol™ column, even at 80% acetonitrile, the Glyburide is retained.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™ 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 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 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
Method Conditions40% 0.05% v/v H3PO4
60% AcetonitrileFlow: 1ml/mnColumn: 75 x 4.6mm idUV: 255nm
Cogent UDC-Cholesterol™bonded
TYPE-C Silica™un-bonded
1 2 3
1 2 3
4
4
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
Cogent UDC-Cholesterol™bonded
TYPE-C Silica™un-bonded
1
1
2
2
3
3
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
Cogent UDC-Cholesterol™bonded
TYPE-C Silica™ un-bonded
1
1
2
2
3
3
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: 255
1- OXALIC ACID2- TARTARIC ACID3- FORMIC ACID4- ACETIC ACID5- SODIUM AZIDE6- URACIL7- FUMARIC ACID8- PROPIONIC ACID
After 3 hours of 100% aqueous exposure, results are the same as with the first injection
1
2
3
4
5
6
7
8
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% Water w/0.05% v/v H3PO4
Flow Rate:1ml/mnColumn: 75 x 4.6mm idUV: 255
1
2 3
4
5
6
7
After 3 hours of 100% aqueous exposure, results are the same as with the first injection
WATER SOLUBLE VITAMINS ON WATER SOLUBLE VITAMINS ON COGENT BIDENTATE C18™ WITH TYPE-C SILICA ™COGENT BIDENTATE C18™ WITH TYPE-C SILICA ™
Method Conditions0-4 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/minColumn: 250 x 4.6mm id
Cogent Bidentate C18UV: 270nm
Peaks Vitamin
LogP1- Niacinamide B3 - 0.45 2- Thiamine Mono-Nitrate B1 -
3.43 3- Pyridoxine HCl B6 - 0.564- Folic Acid B11 - 2.815- Riboflavin B2 - 1.05
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
30%-Distilled Water70% -Methanol
RT: 2.4 min
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 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™
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
Why Use 5 Different Phases Why Use 5 Different Phases in Method Development?in Method Development?
Cogent HPS Cogent HPS C18 C18
3mL/min 20mM NH4COOH & NH4OH pH 8.00
(4) Nortriptyline
(6) Amitryptiline
(5) Imipramine
(3) Toluene
(2) Dimethyl Phthalate
(1) PhenolIsocratic SeparationIsocratic Separation
1 2 + 4
5
3 + 6
Cogent HPSCogent HPS Cyano Cyano
3mL/min 20mM NH4COOH & NH4OH pH 8.00
(4) Nortriptyline
(6) Amitryptiline
(5) Imipramine
(3) Toluene
(2) Dimethyl Phthalate
(1) Phenol
Isocratic SeparationIsocratic Separation
1+2+3
4+5+6
Multi-Mode ChromatographyMulti-Mode Chromatography
Cogent HPS C18
Cogent HPS Cyano
Coupler
Multi-Mode w/ Cogent HPSMulti-Mode w/ Cogent HPS C18 and CyanoC18 and Cyano
3mL/min 20mM NH4COOH & NH4OH pH 8.00
(4) Nortriptyline
(6) Amitryptiline
(5) Imipramine
(3) Toluene
(2) Dimethyl Phthalate
(1) Phenol
Isocratic SeparationIsocratic Separation
(1)
(2)
(3)
(4) (6)
(5)
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