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High Performance Liquid Chromatography ( HPLC ) Primer

HPLC Column Hardware

Design

The goal of the column is to contain the chromatographic packing material (stationary phase), which will be used to effect the separation. The basic design criteria for columns involve the ability to withstand back pressure, properly contain the particles, provide a well-controlled flow path for the sample and individual compound “bands”, and be chemically inert relative to the separation. Typical materials of construction are stainless steel, “PEEK,” an engineered plastic, and glass.

Using a glass column, in which the flow has been stopped part way through the separation, you can see the three dye bands as they separate proceeding down the column.

Separation Performance

The Overall Separation Power of an HPLC consists of 2 main factors; the Mechanical Separation Power, and the Chemical Separation Power.

Resolution

When two compounds are separated, this is called chromatographic “Resolution” (RS). Two “Peaks” would appear in our chromatogram. This Resolution is a function of the Mechanical Separation Power created by the column length and particle size, as well as the Chemical Separation Power created by the “competition” for compounds between the chemistry of the packing material and the mobile phase.

Mechanical Separation Power

The “Mechanical Separation Power is determined by the column length and the particle size. For a given chromatographic particle size, more mechanical separation power (“Efficiency”, “Plate Count”, N) is obtained in longer column lengths, however, the trade-offs are longer chromatographic run times, more solvent consumption and higher back pressure. Shorter column lengths reduce run time and back pressure at the expense of reducing the mechanical separation power.

For a given particle chemistry and mobile phase, a column of the same length, but with a smaller particle size will provide more mechanical separation power. However, the back pressure will increase.

Chemical Separation Power

The choice of the particle chemistry and the mobile phase composition will determine the Chemical Separation Power. There are many different combinations which a scientist may employ in order to create a separation of any two specific compounds. This is discussed in the next section entitled “HPLC Chemical Separation Mechanisms.