HPS™ C18
Method Development Users Guide
Use this guide to predict your sample’s retention characteristics and elution time on the Cogent HPS™ C18 HPLC Column. Included are several examples of different classes of compounds of known Octanol/Water Partition Coefficients (polarity). You can easily determine which pH would be best to use for each of these compound classes with this column. This User Guide will enable you to predict where your compounds should elute for acids, bases and neutrals at three different pH’s.
How to Use This Guide Step 1: Simply compare the Octanol/Water Partition Coefficient of your acidic, basic or neutral compounds with the appropriate compounds shown. Step 2: Read the pH condition at which you will have the best separation with the Generic, LCMS/compatible mobile phase for each compound with this column. Step 3: Predict the optimal pH for your mixture of compounds based on the “retention maps” to resolve your complex mixtures of acids, bases or neutral compounds Octanol/Water Partition Coefficients (Log P) tend to be large for compounds with extended non-polar structures (such as long chain or multi ring hydrocarbons) and small for compounds with highly polar groups. Log P values provides a measure of hydrophilic vs. hydrophobic nature of your compound HPS™ C18
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RETENTION MAP
Acids
11
GRAPH ONE pH 2.18 A: 0.05% H3PO4 B: 100% ACN Acids 1 — Uracil 2 — 4-Hydroxy Benzoic Acid 3 — Acetylsalicylic Acid 4 — 2-Hydroxy Benzoic Acid
11
GRAPH TWO pH 6.80 A: 20mM NH4C00H B: 100% ACN Acids 1 — Uracil 2 — 4-Hydroxy Benzoic Acid 3 — Acetylsalicylic Acid 4 — 2-Hydroxy Benzoic Acid
11
GRAPH THREE pH 8.00 A: 20mM NH4C00H B: 100% ACN Acids 1 — Uracil 2 — 4-Hydroxy Benzoic Acid 3 — Acetylsalicylic Acid 4 — 2-Hydroxy Benzoic Acid
100 90 80 70 60
%B 50 Uracil (1)
40 30 20 10 0
1
2
3
4
5 6 7 RETENTION TIME
8
9
10
Solvent Front Marker 100 90 80
4-Hydroxy Benzoic Acid (2)
70 60
%B 50 40
1234
30 20 10 0
Acetylsalicylic Acid (3)
1
2
3
4
5 6 7 RETENTION TIME
8
9
10
100 90 80 70 60
%B 50 40
1234
30 20
2-Hydroxy Benzoic Acid (4)
10 0
1
2
3
4
5 6 7 RETENTION TIME
8
9
10
Octanol/Water Partition Coefficient Uracil (1) 4-Hydroxy Benzoic Acid (2) Acetylsalicylic Acid (3) 2-Hydroxy Benzoic Acidl (4)
-0.87 1.39 1.13 2.24
Octanol/Water Partition Coefficients (Log P) tend to be large for compounds with extended non-polar structures (such as long chain or multi ring hydrocarbons) and small for compounds with highly polar groups. Log P values provides a measure of hydrophilic vs. hydrophobic nature of your compound HPS™ C18
MicroSolv Technology Corporation 1-732-229-3400 • www.MicroSolvTech.com •
[email protected]
RETENTION MAP
Bases GRAPH FOUR pH 2.18 A: 0.05% H3PO4 B: 100% ACN Bases 1 — Nortryptiline 2 — Imipramine 3 — Amitryptiline
100 90 80 70 60
%B 50 Nortriptyline (1)
40 30 20 10 0
1
2
3
4
5 6 7 RETENTION TIME
8
9
10
11
GRAPH FIVE pH 6.80 A: 20mM NH4C00H B: 100% ACN Bases 1 — Nortryptiline 2 — Imipramine 3 — Amitryptiline
100 90 80 70
Imipramine (2)
%B
60 50 40 30 20 10 0
1
2
3
4
5 6 7 RETENTION TIME
8
9
10
11
GRAPH SIX pH 8.00 A: 20mM NH4C00H B: 100% ACN Bases 1 — Nortryptiline 2 — Imipramine 3 — Amitryptiline
100 90
Amitriptyline (3)
80 70 60
%B 50 40 30 20 10 0
1
2
3
4
5 6 7 RETENTION TIME
8
9
10
11
Octanol/Water Partition Coefficient Nortriptyline (1) Imipramine (2) Amitriptyline (3)
1.63 3.49 2.18
Octanol/Water Partition Coefficients (Log P) tend to be large for compounds with extended non-polar structures (such as long chain or multi ring hydrocarbons) and small for compounds with highly polar groups. Log P values provides a measure of hydrophilic vs. hydrophobic nature of your compound HPS™ C18
MicroSolv Technology Corporation 1-732-229-3400 • www.MicroSolvTech.com •
[email protected]
RETENTION MAP
Neutrals & DMP* GRAPH SEVEN pH 2.18 A: 0.05% H3PO4 B: 100% ACN Neutrals 1 — Uracil 2 — Dimethyl Phthalate 3 — Toluene 4 — Biphenyl 5 — Phenantharene
100 90 80 70 60
Uracil (1)
%B 50 40 30 20 10 0
Solvent Front Marker
1
2
3
4
5
6
7
8
9
10
11
RETENTION TIME
GRAPH EIGHT pH 6.80 A: 20mM NH4C00H B: 100% ACN Neutrals 1 — Uracil 2 — Dimethyl Phthalate 3 — Toluene 4 — Biphenyl 5 — Phenantharene
100 90
Dimethyl Phthalate (2)
80 70 60
%B 50 40 30 20 10
Toluene (3)
0
1
2
3
4
5 6 7 RETENTION TIME
8
9
10
11
GRAPH NINE pH 8.00 A: 20mM NH4C00H B: 100% ACN Neutrals 1 — Uracil 2 — Dimethyl Phthalate 3 — Toluene 4 — Biphenyl 5 — Phenantharene
100 90 80 70
%B Biphenyl (4)
60 50 40 30 20 10 0
1
2
3
4
5
6
7
8
9
10
11
RETENTION TIME
Phenanthrene (5)
Octanol/Water Partition Coefficient Octanol/Water Partition Coefficients (Log P) tend to be Uracil (1) -0.87 large for compounds with extended non-polar structures Dimethyl Phthalate (2) 1.66 (such as long chain or multi ring hydrocarbons) and small for compounds with highly polar groups. Log P values proToluene (3) 2.54 vides a measure of hydrophilic vs. hydrophobic nature of your compound HPS™ C18 Biphenyl (4) 3.76 Phenanthrene (5) 4.35 * Added to mixture. Acts like a Neutral Compound.
MicroSolv Technology Corporation 1-732-229-3400 • www.MicroSolvTech.com •
[email protected]
