can your hplc column do this ?
DESCRIPTION
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. Flow rate: 1ml/mn Column: 75 x 4.6 mm id Isocratic - PowerPoint PPT PresentationTRANSCRIPT
![Page 1: CAN YOUR HPLC COLUMN DO THIS ?](https://reader036.vdocuments.site/reader036/viewer/2022062309/56813c08550346895da56b42/html5/thumbnails/1.jpg)
CAN YOUR HPLC COLUMN DO THIS ?CAN YOUR HPLC COLUMN DO THIS ?
![Page 2: CAN YOUR HPLC COLUMN DO THIS ?](https://reader036.vdocuments.site/reader036/viewer/2022062309/56813c08550346895da56b42/html5/thumbnails/2.jpg)
Type B SILICA TYPE-C SILICA Type B SILICA TYPE-C SILICA
![Page 3: CAN YOUR HPLC COLUMN DO THIS ?](https://reader036.vdocuments.site/reader036/viewer/2022062309/56813c08550346895da56b42/html5/thumbnails/3.jpg)
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
![Page 4: CAN YOUR HPLC COLUMN DO THIS ?](https://reader036.vdocuments.site/reader036/viewer/2022062309/56813c08550346895da56b42/html5/thumbnails/4.jpg)
![Page 5: CAN YOUR HPLC COLUMN DO THIS ?](https://reader036.vdocuments.site/reader036/viewer/2022062309/56813c08550346895da56b42/html5/thumbnails/5.jpg)
COGENT UDC-CHOLESTEROLCOGENT UDC-CHOLESTEROL w/ TYPE-C SILICA w/ TYPE-C SILICA
![Page 6: CAN YOUR HPLC COLUMN DO THIS ?](https://reader036.vdocuments.site/reader036/viewer/2022062309/56813c08550346895da56b42/html5/thumbnails/6.jpg)
Cogent UDC-CholesterolCogent UDC-Cholesterol on TYPE-C Silica™on TYPE-C Silica™
![Page 7: CAN YOUR HPLC COLUMN DO THIS ?](https://reader036.vdocuments.site/reader036/viewer/2022062309/56813c08550346895da56b42/html5/thumbnails/7.jpg)
Cogent HPS C18 Cogent UDC-Cholesterol
Flow Rate: 3ml/min
Wavelength: 254nm
Solvents A: Buffer B: 100%ACN
Neutral CompoundsNeutral Compounds
![Page 8: CAN YOUR HPLC COLUMN DO THIS ?](https://reader036.vdocuments.site/reader036/viewer/2022062309/56813c08550346895da56b42/html5/thumbnails/8.jpg)
Cogent HPS C18 Cogent UDC-Cholesterol
Flow Rate: 3ml/min
Wavelength: 254nm
Solvents A: Buffer B: 100%ACN
Acidic CompoundsAcidic Compounds
![Page 9: CAN YOUR HPLC COLUMN DO THIS ?](https://reader036.vdocuments.site/reader036/viewer/2022062309/56813c08550346895da56b42/html5/thumbnails/9.jpg)
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
![Page 10: CAN YOUR HPLC COLUMN DO THIS ?](https://reader036.vdocuments.site/reader036/viewer/2022062309/56813c08550346895da56b42/html5/thumbnails/10.jpg)
Phenol-Pyridine CompoundsPhenol-Pyridine CompoundsCogent HPS C18 Cogent UDC-Cholesterol
Flow Rate: 3ml/min
Wavelength: 254nm
Solvents A: Buffer B: 100%ACN
![Page 11: CAN YOUR HPLC COLUMN DO THIS ?](https://reader036.vdocuments.site/reader036/viewer/2022062309/56813c08550346895da56b42/html5/thumbnails/11.jpg)
Cogent HPS C18 Cogent UDC-Cholesterol
Flow Rate: 3ml/min
Wavelength: 254nm
Solvents A: Buffer B: 100%ACN
Basic CompoundsBasic Compounds
![Page 12: CAN YOUR HPLC COLUMN DO THIS ?](https://reader036.vdocuments.site/reader036/viewer/2022062309/56813c08550346895da56b42/html5/thumbnails/12.jpg)
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
![Page 13: CAN YOUR HPLC COLUMN DO THIS ?](https://reader036.vdocuments.site/reader036/viewer/2022062309/56813c08550346895da56b42/html5/thumbnails/13.jpg)
RETENTION TIME MAP URETENTION TIME MAP UDC-CHOLESTEROLDC-CHOLESTEROL 150 x 4.6 mm ID
![Page 14: CAN YOUR HPLC COLUMN DO THIS ?](https://reader036.vdocuments.site/reader036/viewer/2022062309/56813c08550346895da56b42/html5/thumbnails/14.jpg)
REAL LIFE UNIQUE APPLICATION:REAL LIFE UNIQUE APPLICATION:
METFORMIN GLYBURIDE
THE CHALLENGE:Separate: LogP of –2.64 & +4.79… Isocratically
![Page 15: CAN YOUR HPLC COLUMN DO THIS ?](https://reader036.vdocuments.site/reader036/viewer/2022062309/56813c08550346895da56b42/html5/thumbnails/15.jpg)
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?
![Page 16: CAN YOUR HPLC COLUMN DO THIS ?](https://reader036.vdocuments.site/reader036/viewer/2022062309/56813c08550346895da56b42/html5/thumbnails/16.jpg)
Let us review…
Un-bonded Un-bonded Type-C SilicaType-C Silica™™ compared to
Cogent-Cogent-UDC CholesterolUDC Cholesterol™™
![Page 17: CAN YOUR HPLC COLUMN DO THIS ?](https://reader036.vdocuments.site/reader036/viewer/2022062309/56813c08550346895da56b42/html5/thumbnails/17.jpg)
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
![Page 18: CAN YOUR HPLC COLUMN DO THIS ?](https://reader036.vdocuments.site/reader036/viewer/2022062309/56813c08550346895da56b42/html5/thumbnails/18.jpg)
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…
![Page 19: CAN YOUR HPLC COLUMN DO THIS ?](https://reader036.vdocuments.site/reader036/viewer/2022062309/56813c08550346895da56b42/html5/thumbnails/19.jpg)
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!
![Page 20: CAN YOUR HPLC COLUMN DO THIS ?](https://reader036.vdocuments.site/reader036/viewer/2022062309/56813c08550346895da56b42/html5/thumbnails/20.jpg)
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.
![Page 21: CAN YOUR HPLC COLUMN DO THIS ?](https://reader036.vdocuments.site/reader036/viewer/2022062309/56813c08550346895da56b42/html5/thumbnails/21.jpg)
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.
![Page 22: CAN YOUR HPLC COLUMN DO THIS ?](https://reader036.vdocuments.site/reader036/viewer/2022062309/56813c08550346895da56b42/html5/thumbnails/22.jpg)
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.
![Page 23: CAN YOUR HPLC COLUMN DO THIS ?](https://reader036.vdocuments.site/reader036/viewer/2022062309/56813c08550346895da56b42/html5/thumbnails/23.jpg)
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.
![Page 24: CAN YOUR HPLC COLUMN DO THIS ?](https://reader036.vdocuments.site/reader036/viewer/2022062309/56813c08550346895da56b42/html5/thumbnails/24.jpg)
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
![Page 25: CAN YOUR HPLC COLUMN DO THIS ?](https://reader036.vdocuments.site/reader036/viewer/2022062309/56813c08550346895da56b42/html5/thumbnails/25.jpg)
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
![Page 26: CAN YOUR HPLC COLUMN DO THIS ?](https://reader036.vdocuments.site/reader036/viewer/2022062309/56813c08550346895da56b42/html5/thumbnails/26.jpg)
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
![Page 27: CAN YOUR HPLC COLUMN DO THIS ?](https://reader036.vdocuments.site/reader036/viewer/2022062309/56813c08550346895da56b42/html5/thumbnails/27.jpg)
WHAT ABOUTWHAT ABOUT PHASE COLLAPSE PHASE COLLAPSE
WITH WITH 100% AQUEOUS ON 100% AQUEOUS ON
TYPE-C™ PRODUCTS?TYPE-C™ PRODUCTS?
![Page 28: CAN YOUR HPLC COLUMN DO THIS ?](https://reader036.vdocuments.site/reader036/viewer/2022062309/56813c08550346895da56b42/html5/thumbnails/28.jpg)
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
![Page 29: CAN YOUR HPLC COLUMN DO THIS ?](https://reader036.vdocuments.site/reader036/viewer/2022062309/56813c08550346895da56b42/html5/thumbnails/29.jpg)
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
![Page 30: CAN YOUR HPLC COLUMN DO THIS ?](https://reader036.vdocuments.site/reader036/viewer/2022062309/56813c08550346895da56b42/html5/thumbnails/30.jpg)
WHAT ABOUTWHAT ABOUT PEPTIDES? PEPTIDES?
![Page 31: CAN YOUR HPLC COLUMN DO THIS ?](https://reader036.vdocuments.site/reader036/viewer/2022062309/56813c08550346895da56b42/html5/thumbnails/31.jpg)
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
![Page 32: CAN YOUR HPLC COLUMN DO THIS ?](https://reader036.vdocuments.site/reader036/viewer/2022062309/56813c08550346895da56b42/html5/thumbnails/32.jpg)
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
![Page 33: CAN YOUR HPLC COLUMN DO THIS ?](https://reader036.vdocuments.site/reader036/viewer/2022062309/56813c08550346895da56b42/html5/thumbnails/33.jpg)
SO WHICH SO WHICH TYPE-C™ PHASE TYPE-C™ PHASE
SHOULD I CHOOSE?SHOULD I CHOOSE?
![Page 34: CAN YOUR HPLC COLUMN DO THIS ?](https://reader036.vdocuments.site/reader036/viewer/2022062309/56813c08550346895da56b42/html5/thumbnails/34.jpg)
THE ANSWER IS A THE ANSWER IS A SCOUT KIT ™ SCOUT KIT ™
![Page 35: CAN YOUR HPLC COLUMN DO THIS ?](https://reader036.vdocuments.site/reader036/viewer/2022062309/56813c08550346895da56b42/html5/thumbnails/35.jpg)
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™
![Page 36: CAN YOUR HPLC COLUMN DO THIS ?](https://reader036.vdocuments.site/reader036/viewer/2022062309/56813c08550346895da56b42/html5/thumbnails/36.jpg)
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
![Page 37: CAN YOUR HPLC COLUMN DO THIS ?](https://reader036.vdocuments.site/reader036/viewer/2022062309/56813c08550346895da56b42/html5/thumbnails/37.jpg)
Multi-Mode ChromatographyMulti-Mode Chromatography
Cogent HPS C18
Cogent HPS Cyano
Coupler
![Page 38: CAN YOUR HPLC COLUMN DO THIS ?](https://reader036.vdocuments.site/reader036/viewer/2022062309/56813c08550346895da56b42/html5/thumbnails/38.jpg)
Multi-Mode ChromatographyMulti-Mode Chromatography
• Unlike Mixed Mode Chromatography
• Multi-Mode is reproducible…every time
![Page 39: CAN YOUR HPLC COLUMN DO THIS ?](https://reader036.vdocuments.site/reader036/viewer/2022062309/56813c08550346895da56b42/html5/thumbnails/39.jpg)
Cogent Scout Kits™ Provide:Cogent Scout Kits™ Provide:
SpeedSelectivity ChoicesVersatilityMulti-Mode™ OptionMultiple Column Size Choices for LCMS