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HPLC Application Sheet
Using an Inverse Gradient Technique to Determine Best HPLC Mode: Analysis of Metformin
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printable Application Sheet
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Note: Due to the gradient conditions you might observe fronting of the peak for the basic polar compound as in Figure I.
This is expected. When using an MS as a detector a higher percentage of formic acid can be used than with UV. For UV
detection a higher percent of acid will cause the distortion of the baseline due to the difference in refraction indices
between solvent A and solvent B in gradient analysis. |
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Method Conditions
| Column |
Cogent Bidentate C18™, 4µm, 100A |
| Catalog No. |
40018-75P |
| Dimensions |
4.6 x 75mm |
| Mobile Phase |
A: DI water + 0.1% formin acid
B: Acetonitrile
Inverse Gradient
| Time (min.) |
%A |
%B |
| 0.00 |
0.00 |
100.00 |
| 0.00-15.00 |
50.0 |
50.0 |
| 15.00-20.00 |
50.0 |
50.0 |
| 20.00-20.01 |
0.00 |
100.00 |
| 20.01-25.00 |
0.00 |
100.00 |
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| Flow rate |
1.0 mL/minute |
| Injection Volume |
1 µL |
| Sample |
I. Metformin 1mg/mL in DI water
II. Toluene 1 µL/mL in acetonitrile
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| Detection |
UV 254 nm |
Discussion
A simple Inverse (ANP) gradient was used to Determine the most useful HPLC Mode for:
I – Basic, polar compound - Metformin
II – Neutral compound – Toluene
Gradients can be used to quickly determine if a compound will be retained in the Aqueous Normal Phase (ANP) mode. From the
gradient profile, one can predict what the retention for a basic polar compound will be in an isocratic mode on the same
column. For example, with Metformin, using 80% acetonitrile and 0.1% formic acid, the retention time is 2.5 minutes
(t0 = 0.7 minutes) when using the same column and flow rate as in the ANP Gradient. This compound is well
retained in ANP. When the gradient is used for neutral compounds they remain in the dead volume; unretained in ANP (see
Figure II). |
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