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ANP HPLC Replaces HILIC - With Benefits

Reference Number: AA-02979 Created: 01/21/2017 12:03 PM Last Updated: 03/01/2017 10:40 AM

Many chromatographers have been given the task to develop HPLC and LCMS methods for polar compounds. Those who have chosen HILIC columns often report their gradient methods are problematic causing them frustration, more method development time and additional costs. Mostly the complaints are the "downtime" between runs that HILIC and all ordinary silica columns struggle with; the equilibration of the on-column, silica water layer. This permanent water layer or "hydration shell" causes difficulty maintaining Precision and reproducibility especially after several runs and when laboratory conditions such as temperature changes. It's also reported that the HILIC column lifetime can be short and the columns can fail without notice during over- night runs. High salt concentration in the mobile phase (ie 100mM) is needed in HILIC  which is a problem for LCMS instruments and preparative chromatography.

ANP or Aqueous Normal Phase HPLC is similar to HILIC in that polar compounds are retained and separated using high organic content mobile phases and running an inverse direction gradient (high organic to high aqueous). One of the differences is that since the Cogent HPLC columns do not retain a water layer (see images below), does not have more than 5% residual silanols and utilizes direct silicon carbon bonds for ligands, the problems most often associate with HILIC are 100% eliminated. Low salt concentrations are used in ANP (ie 15mM or less) and no buffering is needed thus avoiding all the problems of high salt concentrations in LCMS and preparative chromatography.

Some Benefits to the chromatographer using ANP v. HILIC:
  Faster Equilibration between runs (3 column volumes)     Gain more throughput per instrument, save on cost of solvents, 
  Precision is extraordinary run to run, day to day                More confidence in your data, better for GMP and regulated laboratories

  Column Lifetime increased over HILIC columns                Fewer failures during runs, lower cost, Budget saver

  Lower salt needed for retention                                          Does not fowl the inlet of LCMS, faster recovery of prep samples

  ANP can retain polar & some non polar compounds         Wider selectivity range, fast method development

     HILIC Stationary Phases have a water shell           Cogent TYPE-C Silica has no water shell

Summary of Differences Between ANP & HILIC
1. Less than one monolayer of water on Cogent TYPE-C columns v. 3-10 layers on HILIC materials leading to less variability in surface composition and hence greater precision in analyte retention.
2. Rapid equilibration of the stationary phase after gradients, typically in the range of 2-3 columns for Cogent TYPE-C columns.
3. Negatively charged surface on silica hydride due to hydroxide ions.
4. Difference in selectivity and enhanced retention in ANP with Cogent TYPE-C columns.
5. Ability of Cogent TYPE-C columns to function in reversed-phase HPLC mode.
6. Excellent run to run and column to column reproducibility with Cogent TYPE-C columns.
7. No need for high additive concentrations in the mobile phase, only 15mM or less required for any hydrophilic compound analysis.

See also: Main Differences between HILIC & Cogent TYPE-C Columns...
See also: Method Development Guide for ANP
See also: Efficiency of ANP v. HILIC Columns
See also: HILIC v ANP at high flow rate on standard HPLC equipment

For more details on this topic:

C. Kulsing, Y. Nolvachai, P.J. Marriott, R.I. Boysen, M.T. Matyska,
J.J. Pesek, M.T.W. Hearn
 Insights into the Origin of the Separation Selectivity    with Silica Hydride Adsorbents  J. Phys. Chem. B.  2015 119  3063-3069


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