UDC-Cholesterol™
Method Development Users Guide
CHOLESTEROL
Use this guide to predict your sample’s retention characteristics and elution time on the Cogent UDC-Cholesterol Column (150 x 4.6mm ID). 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 and the UDC™ Cholesterol column.
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— — —
Flow Rate: 3ml/min Wavelength: 254 nm
100 90
CHOLESTEROL % ACN
4-Hydroxy Benzoic Acid (1)
80
Solvents A: Buffer B: 100% ACN
70 60
pH 2.18
50 40
— — —
30 20 10 0
1
2
3
Standards
4
5
6
7
8
9
10
11
8
9
10
11
TIME IN MINUTES
1 — 4-Hydroxyl Benzoic Acid 2 — Acetylsalicylic Acid 3 — 2-Hydroxy Benzoic Acid
100 90 80
% ACN
Acetylsalicylic Acid (2)
70 60 50
pH 6.80
40 30 20 10 0
1
2
3
4
5
6
7
TIME IN MINUTES
2-Hydroxy Benzoic Acid (3) 100 90 80
% ACN
— — —
RETENTION MAP
Acids
70 60
pH 8.00
50 40 30 20 10 0
1
2
3
4
5
6
7
8
9
10
11
TIME IN MINUTES
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 and the UDC™ Cholesterol column.
MicroSolv Technology Corporation 1-732-229-3400 • www.MicroSolvTech.com •
[email protected]
90
% ACN
80
Nortriptyline (1)
70
pH 2.18
60 50
30 20 10 0
1
2
3
— — 4 —
Flow Rate: 3ml/min Wavelength: 254 nm Solvents A: Buffer B: 100% ACN
40
5
6
7
8
9
10
11
8
9
10
11
TIME IN MINUTES 100
Standards 1 — Nortriptyline 2 — Imipramine 3 — Amitriptyline
90
Imipramine (2)
% ACN
80 70 60 50 40
pH 6.80
30 20 10 0
1
2
3
4
5
6
7
TIME IN MINUTES 100 90
Amitriptyline (3)
70 60 50 40
▼
Retention Time Reproducibility When cycling pH 8.00 2.00 8.00 1 2 Original 8.3 9.4 First 8.2 9.3 Second 8.2 9.3
80
▼
— — —
100
CHOLESTEROL
% ACN
— — —
RETENTION MAP
Bases
3 9.9 9.8 9.8
pH 8.00
30 20
This requires 15 min equilibration each time.
10 0
1
2
3
4
5
6
7
8
9
10
11
TIME IN MINUTES
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 and the UDC™ Cholesterol column.
MicroSolv Technology Corporation 1-732-229-3400 • www.MicroSolvTech.com •
[email protected]
CHOLESTEROL
90
Dimethyl Phthalate (1)
% ACN
80
Solvents A: Buffer B: 100% ACN
70
pH 2.18
60 50
— — — —
40 30 20 10
0
1
2
3
4
Standards
5
6
7
8
9
10
11
8
9
10
11
8
9
10
11
TIME IN MINUTES
1 — Dimethyl Phthalate 2 — Toluene 3 — Biphenyl 4 — Phenanthrene
100 90
Toluene (2)
80
% ACN
— — — —
Flow Rate: 3ml/min Wavelength: 254 nm
100
70 60 50 40 30
pH 6.80
20 10
Biphenyl (3) 0
1
2
3
4
5
6
7
TIME IN MINUTES 100 90 80
% ACN
— — — —
RETENTION MAP
Neutrals & DMP*
Phenanthrene (4)
70
pH 8.00
60 50 40 30 20 10
0
1
2
3
4
5
6
7
TIME IN MINUTES
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 and the UDC™ Cholesterol column. 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]
