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Development guide for ANP or Aqueous Normal Phase HPLC methods

Reference Number: AA-00417 Created: 04/13/2012 09:15 PM Last Updated: 07/23/2016 03:13 PM

Method Development Guide
Aqueous Normal Phase (ANP) Using Inverse Gradients
 

Ordinary commercial silica media used for HPLC suffers from un-reacted/un-endcapped silanols (hydroxyl groups) which may present un-desirable and/or inconsistent results with respect to high concentrations of polar and some non polar solutes. Cogent TYPE-C Silica™ stationary phases, with their unique silica hydride bonding technology, have the ability to retain polar solutes (at high concentrations) which reside in the mobile phase's organic component as well as some non-polar compounds at the same time. 

The Cogent TYPE-C Silica™ bonded and non bonded phases (Bidentate C18™, Bidentate C8™, UDC-Cholesterol™, Diamond Hydride™, Silica-C™ and Phenyl Hydride™) all possess this capability. The exact point in the composition of the mobile phase where aqueous normal phase (ANP) retention begins depends on the solute as well as the stationary phase selected. Because of the attached organic group that is part of the stationary phase, the TYPE-C Silica™ columns can also retain nonpolar compounds based on the typical reversed phase mechanism. The diagram below illustrates how this dual retention capability works for both polar (metformin) and nonpolar (glyburide) compounds:



A Generic Starting Point for Cogent TYPE-C Columns

It is easy to use a Cogent™ HPLC column that is made with TYPE-C Silica™.

The power of these columns is found in the wide range of solvents that can be used with them and the unique and strong retention they can have for polar compounds while at the same time retaining non polars. Selection of the column (C18, C8, Cholesterol or Silica-C) will be determined by your compounds of interest. Mixtures that will be highly polar and do not contain non polar compounds might be better suited by the Silica-C where compounds with both polar and non polar might be better suited to the Bidentate C18.

Step 1: Mobile Phase Selection. After you have properly installed the column, conditioned it according to our suggestions, it is a good idea to start with a typical gradient run. We suggest starting with an acidified mobile phase of water and acetonitrile. Acidify the mobile phase with up to 0.5% of an acid such as formic or acetic acid. If you are not using LCMS, TFA is another good candidate.

Step 2: Equilibrate (Reversed Phase). Run about 6 column volumes of the mobile phase in Step 1 at 95% water.

Step 3: Reversed Phase Gradient. Set up your instrument to run a shallow gradient from 95% water to 40% water over 20 minutes on a 75mm long column. For longer columns increase the gradient time proportionally. This long and shallow gradient will be very beneficial for determining the optimal gradient or isocratic method to run your mixture with later. For sharper peaks and less retention, run a shorter (Steeper) gradient from the same starting points to end points.

Step 4: Equilibrate (ANP). Equilibrate the column by running 100% acetonitrile for approximately 2 minutes.

Step 5: ANP Gradient. Set up your instrument to run a shallow gradient using the same mobile phase in Step 3 to run from 90% acetonitrile to 40% acetonitrile over 20 minutes for a 75mm long column. For longer columns increase the gradient time proportionally. This long and shallow gradient will be very beneficial for determining the optimal gradient or isocratic method to run your mixture with later. For sharper peaks and less retention, run a shorter (steeper) gradient from the same starting points to end points.

Step 6: Compare the Reversed Phase and ANP Data. Evaluate both gradient runs for retention time, peak shape and elution order as retention on the column is compound specific where some compounds will not retain in Step 3 (Reverse Phase) and some do not retain in Step 5 (Aqueous Normal Phase). One column could produce an isocratic run which retains both polar and non polar compounds.

Note: The Cogent Bidentate C8 and C18 columns have a unique quality in that they sometimes can retain polar compounds not retained on other columns while run at 100% water. You could insert an isocratic run at 100% water after Step 3 and before Step 4.

 

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