shutterstock_1022973487Classical normal phase (NP) and reversed phase (RP) chromatography should never be run on the same HPLC system without system modifications in between. That is an old, golden rule not every LC operator knows about anymore. With high effort, the HPLC system needs to be prepared for either NP or RP operation. This typically includes the exchange of pump seals, certain capillaries and other wetted parts. When done, the HPLC system must be purged thoroughly after a changeover and the entire system downtime is enormously high.

Even though 80% of HPLC applications are (still) RP based, NP applications are essentially needed for the separation of substances which cannot be (sufficiently) separated under RP conditions, typically polar and ionic compounds. As such sample compounds are very common in the biopharma industry, NP gains increasing attention and new importance. However, NP is known for poor retention time reproducibility caused by water or protic organic solvent(s) in the mobile phase or sample solvent which sticks for a long time or forever on the NP stationary phase.

Hexane is not hexane. Cyclohexane becomes solid between 230 and 460 bar (depends on its temperature being 20 or 30 °C) and pumping becomes impossible. It might even be that pump pistons break. Solution: Use n-hexane.

With RP, the stationary phase is nonpolar and the mobile phase is polar, hence a mixture of water (often with additives) and an organic modifier, commonly acetonitrile or methanol. With NP, the stationary phase is polar and the mobile phase is nonpolar; most often hexane or heptane, mixed with a slightly more polar solvent such as isopropanol or ethyl acetate. Furthermore, the mobile phase must be water-free as even traces of water badly affects separation efficiency and result. This is no news and shall just underline that in former times, there was a clear borderline between NP and RP operation.

And nowadays? There is not such a clear differentiation anymore.

Because of the detrimental sensitivity of NP to water traces, hydrophilic interaction liquid chromatography (HILIC) was introduced and still becomes more and more popular. HILIC is also used for the separation of polar and ionic sample compounds. HILIC uses hydrophilic stationary phases (more or less to NP modified RP based columns) with RP type organic eluents and a small amount of aqueous liquid. It is sometimes related to aqueous normal-phase chromatography (ANP), a chromatographic technique that involves the mobile phase region between classical aqueous RP and organic NP. In reality, ANP retention is much more diverse than either simple RP or HILIC behavior. It is an own LC application field which might be – due to its complexity – discussed in another article.

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So far, no problem. RP, HILIC or even ANP applications are run on the same HPLC system. This is also an opportunity for generic LC methods in which RP and HILIC or RP and ANP techniques are coupled in a way that “all” classes of compounds can be adequately separated in a single run. Please refer to the poster note “Generic Method Approach for Pharmaceutical Drug Discovery and Development using Reversed-Phase Hydrophilic Interaction Liquid Chromatography with Universal Charged Aerosol Detection” for more information.

Non-aqueous reversed phase liquid chromatography

Hydrophobic or low polarity substances (such as hydrocarbons or glyceride oils) do not completely or at all elute with classical RP, even when 100% organic solvent is applied on the RP column. Only the addition of lower-polarity solvents such as methylene chloride or tetrahydrofuran to the mobile phase results in rapid elution of these non-polar substances. In other words, an RP column is used, typical NP solvents and, in addition, solvents that are known to have an impact to the lifetime of wetted parts within an HPLC system.

Non-aqueous reversed phase (NARP) is a more and more commonly used technique to separate non-polar compounds with a resolution that is not or at least very difficult to achieve with classical RP, HILIC, NP or ANP applications. NARP is especially suitable for long-chain, non- or weak-polar compounds, e.g. for fast analyses of biodiesel (a mixture of fatty acid esters). Some symmetric molecules such as crown ethers or even regioisomers, e.g. sn-1/3 position fatty acid species or the sn-2 position of triacylglycerols (TAGs) in natural fats and oils, may also be separated.

With the emerging biopharma market, there is also an increasing demand for NARP applications. This may lead to “forgotten” technical problems. This technique uses RP columns but NP solvents, hence an NP compatible HPLC system must be used. This might be a new situation for a lot of users who are not too familiar with classical NP anymore or enjoyed the freedom to run HILIC on their RP system without thinking about system changeover. Now, a changeover from an RP to an NP compatible HPLC system is essential to run NARP. Again, NARP solvents are NP solvents like methanol, ethanol or acetonitrile but also (1) hexane and heptane or (2) methylene chloride or tetrahydrofuran.

Whilst hexane and heptane require NP compatibility of the HPLC system, methylene chloride and tetrahydrofuran typically require more, at least a special modification of the entire HPLC system and the understanding that wear parts have a much lower lifetime than usual, so frequent system downtime must be taken into account. Furthermore, wetted plastics parts (e.g. PEEK) swell; so wear and tear, fatigue or premature ageing of these plastics parts might happen fast. Refer to the system and module manuals and you find a lot of precautions regarding methylene chloride and tetrahydrofuran.

Conclusions

  1. NARP and NP are not RP HPLC system compatible
  2. NARP and NP require a dedicated NP HPLC system
  3. Solvents like methylene chloride or tetrahydrofuran are not system fluidics friendly, so should only be used when really needed