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Study Notes: The Column

In HPLC the column and the mobile phase are the most important components, as it is the interaction of the analyte with each of these that permits separation of the analytes in the sample.

The column is the separating component of the HPLC and basically consists of a steel or compressed plastic tube into which is packed the separation material. Columns usually range in length from 10 to 30 cm (internal diameter 4 to 10 mm) with common packing sizes of 3, 5 and 10 µm). Typically columns contain 40 000 to 60 000 plates/m (N).

High performance columns (internal diameter 1 to 4.6 mm), packed with 3 or 5 µm particles are also available. Such a column would contain about 100 000 plates/m.

High performance columns allow very rapid separations. For instance, eight analytes in a sample may be separated in 30 seconds Chromatogram with 8 peaks, the last of which occurs at 30 seconds.

The column must have the following characteristics.

  • Be rigid and able to withstand very high pressures (up to 10,000 psi [~69,000 kPa]).
  • Be packed with material that is compatible with the sample and the mobile phase.
  • Be packed with material that will cause the separation of the analytes of interest.
  • Be packed with material of the appropriate pore size for the separation.

There are two basic physical forms of column packings:

  • Pellicular - are spherical, nonporous glass or polymer beads (diameter 30 to 40 µm) that are coated with a thin porous layer of silica, alumina or ion-exchange resin. A liquid stationary phase may also be applied. Alternatively, the beads may be chemically treated to produce an organic surface layer. These column packings are mainly used for guard columns and not for analytical purposes.
  • Porous Particle - are uniform porous microparticles (diameter 3 to 10 µm) composed of silica, alumina or ion-exchange resin. Silica is the most common. They are often coated with thin organic films bonded to the surface.

There are four main types of HPLC applications:

  • Adsorption
  • Exclusion
  • Ion-exchange
  • Partition (normal and reversed-phase).

This figure shows the major types of HPLC applications based on molecular weight versus polarity and water solubility of an analyte.

Saunders, D. (1975), Chromatography, New York: Van Nostrand Reinhold, as reproduced in Skoog, D. and Leary, J. (1992), Principles of Instrumental Analysis, Fort Worth: Saunders College Publishing.

Partition HPLC is the most widely used of the four techniques and is routinely used for non-ionic polar compounds of low to moderate Mr of <3 000. Partition chromatography can be subdivided into liquid and bonded-phase chromatography. The difference being in the bonding of support material to the stationary phase with liquid-liquid bonded by physical adsorption, and bonded-phase bound by chemical bonds.

The bonded-phase predominates today and is composed of uniform, porous, sturdy silica to which are bonded siloxanes to form the liquid layer that is the actual stationary phase.

There are two types of partition chromatography based on the relative polarities of the mobile and stationary phases. In normal-phase packings the stationary phase is relatively polar and the mobile phase non-polar (for example, hexane) whilst for reversed-phase packings the stationary phase is non-polar (often a hydrocarbon) and the mobile phase is relatively polar (such as water, methanol, acetonitrile). The terminology is historical, that is, the normal-phase separations were used for early work and the reversed-phase applications came later.

In normal phase chromatography the least polar component is eluted first as it is the most soluble in the mobile phase and increasing the polarity of the mobile phase has the effect of decreasing the elution time.

In reversed-phase methods, the most polar component appears first and increasing the mobile phase polarity increases the elution time.

Diagrammatical peaks showing that normal-phase peaks are in the order 3, 2, 1 whereas reversed-phase peaks are in the order 1, 2, 3. High polarity mobile phase shows separated peaks, while medium polarity mobile phase shows the peaks closer together.

In the diagram above component A is the most polar and component C is the least polar.

Bonded phase packings are considered as in the following.

  • Reversed-phase when the bonded coating is nonpolar in character.
  • Normal-phase when the bonded coating contains polar functional groups.

Reversed-phase packings are used in up to 75% of HPLC separations. Most commonly the active group of the siloxane is a C8 (n-octyl) or a C18 (n-octodecyl) chain.

Reversed-phase HPLC will be used for the examples later in this section but you should be aware that other HPLC systems exist and are used for various applications.

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