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Notes on Using TYPE-C™ HPLC Columns in ANP or Normal Phase modes

When using Reverse Phase (RP) mode for Cogent TYPE-C™ columns, you will normally observe results similar to most Reverse Phase columns in that they are rarely, extremely critical with respect to retention time or relative selectivity to % changes of organic modifier of + or – 1% or of temperature of + or – 1° Celsius.

Using Normal Phase Modes:

This situation is not the same for type Type B columns in organic normal phase (ONP), as the small changes noted above in polar organic content or temperature can drastically effect retention and selectivity. This sensitivity to % of polar organic and temperature is also noted in Cogent TYPE-C Silica™ columns and related phases when operating in aqueous normal phase or organic normal phase.

Temperature changes between 10 and 50° Celsius have been shown to change the elution order of three antidepressants samples. Therefore it is important to precisely control the temperature of any column used in Normal Phase Separation Methods.

Similarly it will be noted that a minor change in percentage from 8 to 10 % aqueous content can significantly increase retention and resolution (relative selectivity) of some compounds.

Practical experience has shown that the criticality of concentration of solvents A to B in Normal Phase modes makes it impractical to develop methods in fine detail when using 100 % A + 100% B and using a conventional HPLC pump gradient mixing (high or low pressure gradient formation).

We advise that the approximate % concentration of solvents are found by this method (mixing pumps), but final % concentrations are defined either by isocratic v/v mixing (note as organic solvents are not additive on volume when mixing, separately measure out each solvent; add, shake and de-gas, the ultimate volume of mix is irrelevant) or change solvent A to a mixture of solvents A + B ( say 20% A in 80% B ) to be called C, thus 10% solvent A becomes a 50:50 mixture of solvents B and C and 5% A, a mix of 75:25 B + C.

The latter situation is one that most modern HPLC pumps can manage, but day to day reproducibility of a mix of 5:95 A + B in ONP and ANP seems impossibly difficult for many gradient HPLC pumps. Check with your system manufacturer for recommendations.

Isocratic premixing and accurate temperature control with a forced air circulation oven/chiller is, however, always preferred in ANP plus ONP be it pre-existing irregular, type A, type B or MicroSolv’s evolutionary Cogent TYPE-C™ normal phase chromatography for reproducible, robust methods.

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