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HPLC Application Sheet
Using an Inverse Gradient Technique to Determine Best HPLC Mode: Analysis of Metformin

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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.


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
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
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).