HPLC
TM
TM
Using Aqueous Normal Phase (ANP) and Aqueous Reverse Phase (ARP) on One TYPE-C™ Based HPLC Column Swap Peak Order… Can Your HPLC Column Do This? Method Conditions
Retention Map
Mobile Phase
Chromatograms A & B Solvent A: Aqueous (0.05% H3P04) Solvent B: Acetonitrile
Column
Cogent UDC-Cholesterol™
Cat. No.
69069-75R
Dimensions
75 x 4.6mm id
100 90
70 60
100
pH 2.18
50 40
90
Standards 1 — Uracil 2 — Pyridine 3 — Phenol
30 20 10
0
1
2
3
4
5
6
7
8
9
10
11
80
% ACN
% ACN
80
70 60 50 40 30 20
TIME IN MINUTES
Chromatogram A: Mobile Phase A -20% B -80%
2
Flow Rate
ml/min pH1.0 2.18
Detection:
UV, 240nm
10
0
SOLVENT FRONT
Standards 1 — Uracil 2 — Pyridine 3 — Phenol
UDC Cholesterol
1
2
3
4
5
6
7
8
9
10
11
RetentionTIME Map: IN MINUTES Useful tool, for quickly developing methods in HPLC: Here, Phenol exhibits a typical reverse phase plot (ARP) while both Uracil and Pyridine are showing normal phase characteristics in aqueous conditions (ANP).
Discussion of Chromatograms 1
1
Chromatograms A & B Peak 1 (Metformin) eluted with longer retention time as the concentration of Acetonitrile was increased. During the same run, Peak 2 (Glyburide) elutes with shorter retention time as concentrations of Acetonitrile are increased.
4
Chromatogram B: 1 SOLVENT FRONT
APPLICATION NOTES
No. 1 of Series D
1
2
Mobile Phase A -50% B -50%
4
Peaks 1: Metformin Log P Minus 2.6 2: Glyburide Log P Plus 4.79
Implications and Advantages Metformin is a very polar pharmaceutical compound which, chromatographically, is very similar to Pyridine (see Retention Map above). Glyburide, a neutral pharmaceutical ingredient is often formulated with Metformin, which is so polar that it elutes on the solvent front on other C18, Cyano, Amino, Phenyl and F5 columns. To quantify these compounds, two runs with different methods were previously necessary. Uses By using one UDC-Cholesterol™ HPLC Column, you can isocratically achieve separation and get confirmation of your peaks with the column’s orthogonal selectivity. Simply modify the mobile phase as shown here to save time and lab resources.
MTC • MicroSolv Technology Corporation 1 Industrial Way West, Bldg E, Eatontown, NJ 07724 USA Ph. 1-732-578-1777 • Fx. 1-732-578-9777 www.mtc-usa.com Email:
[email protected] 03-H-09-D1
