Date: 17-JANUARY-2022   Last Updated: 30-NOVEMBER-2025

Generic Tips for Developing HPLC Methods with Cogent TYPE-C™ Columns

Cogent TYPE-C Silica columns offer exceptional versatility, supporting Aqueous Normal Phase (ANP), Reversed Phase (RP), and Organic Normal Phase (ONP) modes. Their unique surface chemistry provides strong retention for polar compounds while still accommodating non-polar analytes, making them ideal for complex mixtures.

Column Selection:
Choose based on your analytes:

• Highly polar mixtures: Diamond Hydride, Phenyl Hydride, Amide, Silica-C
• Mixed polarity: Bidentate C18, Bidentate C8, UDC-Cholesterol, Phenyl Hydride, Amide

Step-by-Step Method Development

Step 1: Mobile Phase Selection

Start with a gradient run using an acidified water/acetonitrile system:

• Acidify with up to 0.5% formic or acetic acid.
• ​ For non-LC-MS applications, TFA (≤0.01%) is acceptable.

Step 2: RP Equilibration

Run ~6 column volumes of mobile phase at 95% water.

Step 3: RP Gradient

Program a shallow gradient from 95% water → 40% water over 20 min.

• For sharper peaks and reduced retention, use a steeper gradient.

Step 4: ANP Equilibration

Flush with 100% acetonitrile, then equilibrate at 90:10 acetonitrile/DI water for ~5 min .

Step 5: ANP Gradient

Run a shallow gradient from 90% acetonitrile → 40% acetonitrile over 20 min.

• For faster elution, use a steeper gradient.

Step 6: Compare RP vs ANP

Evaluate retention, peak shape, and elution order. Some compounds may only retain in RP or ANP, while others in both—allowing for isocratic runs that handle mixed polarity.

Additional Note: Cogent Bidentate C8 and C18 columns can retain certain polar compounds even at 100% water. Consider adding an isocratic run at 100% water after Step 3 for additional insight.