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Separation Modes

Reversed-Phase Chromatography

Reversed-phase chromatography is by far the most popular liquid chromatography separation technique employed in the laboratory today, and its popularity is reflected by the large selection of column products currently available from Waters.

In its simplest form, reversed-phase chromatography comprises of a polar mobile phase, typically mixtures of water or buffer with polar solvents such as methanol, acetonitrile or tetrahydrofuran, and a non-polar stationary phase such as a long-chain hydrocarbon bonded to a silica or hybrid support. In recent years, significant research and development effort has been targeted in the area of separation media aimed at improving column efficiency, pH stability and providing alternate selectivities.

Modern column chemistries from Waters can be broadly classified into three categories:

  • Hybrid – Designed for maximum pH stability in order to provide the most flexible method development options. Available in XBridge, ACQUITY BEH, and XTerra brands; pH range 1-12.
  • Silica – Historically, the most commonly used reversed-phase support, SunFire™ and Symmetry® products are synthesized from raw materials in Waters Taunton manufacturing facility and are designed for maximum loadability; pH range 2-7
  • Application-specific – Designed to target a specific application area, such as Atlantis® columns for enhanced retention of polar molecules.

Normal-Phase Chromatography

Before the development of reversed-phase bonded phases, normal-phase chromatography was the most popular separation technique. It relies on the interaction of analytes with polar functional groups on the surface of the stationary phase, which is strongest when non-polar solvents are used as the mobile phase.

Normal-phase chromatography is a very powerful separations tool because of the wide range of solvents available that can be used to fine tune the selectivity of a separation. However, it has fallen into disfavor with many chromatographers because of some of the complexities involved. Under some circumstances, lengthy equilibration times or reproducibility problems may be encountered which are due largely to the sensitivity of the technique to the presence of small concentrations of polar contaminants in the mobile phase. If these problems are controlled, the technique typically gives chromatograms superior to reversed-phase methods due to the low viscosity of the commonly used solvents.

Normal-phase columns are available in the SunFire, Nova-Pak and Spherisorb product families.

HILIC (Hydrophilic-Interaction Chromatography)

Hydrophilic-interaction chromatography has been practiced for a long time, but the term HILIC has only recently come into common usage. Analytes are very polar compounds such as polar metabolites, carbohydrates or peptides.

HILIC can be viewed as an extension of normal-phase chromatography into the realm of aqueous mobile phases. The mobile phases are mixtures of water or buffer (< 40%) with organic solvents. The stationary phases are very hydrophilic polar adsorbants such as silica, polar bonded phases, polar polymeric packings, and ion exchangers. The common factor of all of these stationary phases is that they can easily adsorb water, hence the categorization of “hydrophilic”.

Gradient methods employed in HILIC mode are exactly the opposite of those in reversed-phase mode. Initial conditions will comprise of high organic, typically 95% moving progressively to higher aqueous. For this reason, the term reversed-reversed-phase is also becoming common.

The technique is gaining in popularity and for this reason is the subject of increasing column packings research.

Waters HILIC columns are currently available in the Atlantis, CORTECS, XBridge and ACQUITY product families.