Transcript TYPE-C™ HPLC Columns PowerPoint Presentation
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 Uracil & Phenol Toluene Biphenyl Uracil Phenol Toluene Biphenyl
Flow rate: 1ml/mn Column: 75 x 4.6mm id Isocratic: 60% - 0.04% v/v
H 3 PO 4
40%ACN
Type B Silica TYPE C Silica™
Cogent UDC-Cholesterol
on TYPE C Silica™
Cogent UDC-Cholesterol
on TYPE C Silica™
Cogent HPS
Neutral Compounds
C18 Cogent UDC-Cholesterol
Flow Rate
: 3ml/min
Wavelength
: 254nm
Solvents
A: Buffer B: 100%ACN
Cogent HPS
Acidic Compounds
C18 Cogent UDC-Cholesterol
Flow Rate
: 3ml/min
Wavelength
: 254nm
Solvents
A: Buffer B: 100%ACN
Cogent UDC-Cholesterol
on TYPE C Silica™
Phenol & Pyridine Neutral Acid
Peak Order Peak Order Peak Order pH 8.00 pH 2.18
pH 2.18
5% 20mM NH 4 COOH/NH 4 OH 60% (H 3 PO 4 ) 90% (H 3 PO 4 ) 95% Acetonitrile 40% Acetonitrile 10% Acetonitrile
Phenol-Pyridine Compounds
Cogent HPS
C18 Cogent UDC-Cholesterol
Flow Rate
: 3ml/min
Wavelength
: 254nm
Solvents
A: Buffer B: 100%ACN
Cogent HPS
Basic Compounds
C18 Cogent UDC-Cholesterol
Flow Rate
: 3ml/min
Wavelength
: 254nm
Solvents
A: Buffer B: 100%ACN
Cogent UDC-Cholesterol
on TYPE C Silica™ Normal Phase Resolution of Basic Antidepressants
Solvents: A: 10% B: 90% Solvents: A: 8% B: 92%
Solvent A: 20mM NH 4 COOH/NH Solvent B: Acetonitrile 4 OH
pH 8.00
RETENTION TIME MAP UDC-CHOLESTEROL
Showing Both Normal and Reverse Phase Utility
Column Dimensions 150x4.6mm
REAL LIFE, UNIQUE APPLICATION:
METFORMIN GLYBURIDE THE CHALLENGE : Separate: LogP of – 2.64 & + 4.79… Isocratically
COGENT UDC-CHOLESTEROL
w/ TYPE-C SILICA
A: 99.95% Aqueous - 0.05% H 3 PO 4 (pH 2.18) B: 99.95% Acetonitrile - 0.05% H 3 PO 4 Flow Rate: Column: 1 ml/min 75 x 4.6mm id UV : 210nm Back pressure: 600 psi Metformin log P = -2.64
Glyburide log P = +4.79
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?
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 = MeOH A: 80% B: 20% AQUEOUS Reverse Phase = ACN A: 90% E: 10% A: 0.05%v/v H 3 PO 4 (pH 2.15) B: MeOH C: Acetonitrile D: THF E: Hexane F: Ethyl Acetate AQUEOUS Reverse Phase = THF A: 90% D: 10% ORGANIC Normal Phase = Hexane & Ethyl Acetate E: 95% F: 5% Flow Rate: 1ml/min Column: 75 x 4.6mm id UV: 255nm
TYPE-C SILICA ™ CAN SEPARATE
METFORMIN
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
Glyburide
HOWEVER…
COGENT UDC-CHOLESTEROL
w/ TYPE-C SILICA
A: 99.95% Aqueous - 0.05% H 3 PO 4 (pH 2.18) B: 99.95% Acetonitrile - 0.05% H 3 PO 4 Flow Rate: Column: 1 ml/min 75 x 4.6mm id UV : 210nm Back pressure: 600 psi Metformin log P = -2.64
Glyburide log P = +4.79
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
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… 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?
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?
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 Silica™ - unbonded column.
from that of the TYPE-C 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 Cogent UDC-Cholesterol ™
bonded 1 2 3 4
Method Conditions 40% 0.05% v/v H 3 PO 4 60% Acetonitrile Flow: 1ml/mn Column: 75 x 4.6mm id UV: 255nm TYPE-C Silica ™
un-bonded 1 2 3 4
Method Conditions 60% 0.05% v/v H 3 PO 4 40% Acetonitrile Flow: 1ml/mn Column: 75 x 4.6mm id UV: 255nm
SELECTIVITY COMPARISON IN AQUEOUS REVERSE PHASE OF URACIL, PYRINDINE, PHENOL Cogent UDC-Cholesterol ™
bonded
Method Conditions 80% 0.05% v/v H 3 PO 4 20% Acetonitrile Flow: 1ml/mn Column: 75 x 4.6mm id UV: 255nm
1 2 3
TYPE-C Silica ™
un-bonded 1 2 3
Method Conditions 90% 0.05% v/v H 3 PO 4 10% Acetonitrile Flow: 1ml/mn Column: 75 x 4.6mm id UV: 255nm
SELECTIVITY COMPARISON IN AQUEOUS REVERSE PHASE OF AROMATIC ORGANIC ACIDS Cogent UDC-Cholesterol ™
bonded
Method Conditions 80% 0.05% v/v H 3 PO 4 20% Acetonitrile Flow: 1ml/mn Column: 75 x 4.6mm id UV: 255nm
1 2 3
TYPE-C Silica ™
un-bonded 1 2 3
Method Conditions 90% 0.05% v/v H 3 PO 4 10% Acetonitrile Flow: 1ml/mn Column: 75 x 4.6mm id UV: 255nm
WHAT ABOUT PHASE COLLAPSE WITH 100% AQUEOUS ON TYPE C™ PRODUCTS?
USING 100 % AQUEOUS ON COGENT UDC CHOLESTEROL™ WITH TYPE-C SILICA™ 3 1 2 4 6 7 8 After 3 hours of 100% aqueous exposure, results are the same as with the first injection 1 - OXALIC ACID 2 - TARTARIC ACID 3 - FORMIC ACID 4 - ACETIC ACID 5 - SODIUM AZIDE 6 - URACIL 7 - FUMARIC ACID 8 - PROPIONIC ACID 5 100% 0.05% v/v H 3 PO 4 Flow Rate: 1ml/mn Column: 75 x 4.6mm id UV: 255
USING 100 % AQUEOUS ON pH STABLE COGENT BIDENTATE C18™ WITH TYPE-C SILICA™ 1 4 6 After 3 hours of 100% aqueous exposure, results are the same as with the first injection 1 - OXALIC ACID 2 - FORMIC ACID 3 - ACETIC ACID 4 - SODIUM AZIDE 5 - URACIL 6 - FUMARIC ACID 7 - PROPIONIC ACID 2 3 5 7 100% Water w/0.05% v/v H 3 PO 4 Flow Rate:1ml/mn Column: 75 x 4.6mm id UV: 255
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 10-15 min: Equilibrate start conditions Flow Rate: 1ml/min Column: 250 x 4.6mm id UV: Cogent Bidentate C18 270nm Peaks Vitamin LogP 1- Niacinamide B3 2- Thiamine Mono-Nitrate B1 - 0.45 - 3.43
3- Pyridoxine HCl B6 - 0.56
4- Folic Acid B11 - 2.81
5- Riboflavin B2 - 1.05
WHAT ABOUT PEPTIDES?
PEPTIDE ANALYSIS ON COGENT UDC CHOLESTEROL™
RT: 3.6 min 30%-Distilled Water 70% -Methanol
Peptide 6 11 Amino Acid Residues Centered around Aspargine Flow Rate: 0.5ml/mn Column: 150 x 4.6mm id UV: 210nm
RT: 2.4 min 20%-Distilled Water 70% -Methanol
Peptide 9 11 Amino Acid Residues Centered around Aspargine
PEPTIDE ANALYSIS ON COGENT UDC CHOLESTEROL™
RT: 3.7 min.
30%- Distilled Water 70%- Methanol
Peptide 11 9 Amino Acid Residues Centered around Histidine
RT: 2.4 min.
10%- Distilled Water 90%- Methanol
Flow Rate: 0.5ml/mn Column: 150 x 4.6mm id UV: 210nm Peptide 11 9 Amino Acid Residues Centered around Histidine
SO WHICH TYPE C™ PHASE SHOULD I CHOOSE?
THE ANSWER IS A SCOUT KIT ™
Cogent Scout Kits™
150mm x 4.6mm ID 75mm x 4.6mm ID 50mm x 3.0mm ID 50mm x 2.1mm ID 20mm x 4.0mm ID 20mm x 2.0mm ID Scout Kit™ Mini Scout Kit™ LC MS Methods Kit™ LC MS Methods Kit™ Ballistic LC MS Kit™ Mini 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
Isocratic Separation
1 2 + 4 5
(1) Phenol (2) Dimethyl Phthalate (3) Toluene
3 + 6
(4) Nortriptyline (5) Imipramine (6) Amitryptiline 3mL/min 20mM NH 4 COOH & NH 4 OH pH 8.00
Cogent HPS
Cyano
Isocratic Separation
(1) Phenol ( 2) Dimethyl Phthalate
1+2+3
(3) Toluene
4+5+6
(4) Nortriptyline (5) Imipramine (6) Amitryptiline 3mL/min 20mM NH 4 COOH & NH 4 OH pH 8.00
Multi-Mode Chromatography
Cogent HPS
C18 Coupler Cogent HPS
Cyano
Multi-Mode w/ Cogent HPS C18 and Cyano Isocratic Separation
(1) Phenol (2) Dimethyl Phthalate
(1) (4) (5) (6)
(3) Toluene
(2)
(4) Nortriptyline
(3)
(5) Imipramine (6) Amitryptiline 3mL/min 20mM NH 4 COOH & NH 4 OH pH 8.00
Multi-Mode Chromatography
• Unlike Mixed Mode Chromatography • Multi Mode is reproducible…every time
Cogent Scout Kits™ Provide:
Speed
Selectivity Choices
Versatility
Multi Mode™ Option