# Performance Testing of Packed Columns

# Guidelines for Performance Testing of Packed Columns

To check the quality of the column packing, the** theoretical number of plates** and **peak
asymmetry **must be established. This is achieved by quantifying the geometry of an isocratic peak, which can be produced by injection of a marker detectable by spectrophotometry, pH or

conductivity.

For hydrophobic ligands, a solution such as 2 M NaCl should be used. NaCl will not interact

with the ligand chemistry in such a way as to cause an anomalous result. Otherwise a solution

of 2 % (v/v) acetone may be used. The injection volume should be 1 – 5 % of the column

volume. For the mobile phase a 0.1 M NaCl solution is recommended to be used.

Number of plates can be calculated using the following equation:

**N = 5.54 (V _{e}/W_{1/2})^{2}**

where:

N = number of theoretical plates

V_{e} = elution volume/retention time of marker molecule

W_{1/2} = width of peak at half its maximum deflection

Refer to the graph below for illustration of these parameters. For the calculation to work, the time and width values must both be reported as the same units of measure: geometric lengths observed on the chromatogram, or the time observed for both events to occur.

To allow comparison between different chromatography adsorbents, it is useful to determine the **Reduced plate height (h)**

h = N/dp

where:

N = number of theoretical plates

dp = diameter of the adsorbent bead

**Asymmetry** can be calculated as follows:

A_{s} = b/a

where:

a = the distance from the leading edge of the peak to the mid-point of the peak

b = the distance from the mid-point of the peak to trailing edge.

As is measured using data at 10% of the maximum peak height.

Whilst it is difficult to give exact parameters a column with a reduced plate height (h) of between 1.5 to 2 and an asymmetry factor between 0.8 < A_{S} < 1.8 would be acceptable when using porous chromatography media in a bioprocess application.

An asymmetry value of < 0.8 indicated an **over packed** column and the column should be repacked using a reduced flow and/or pressure

An asymmetry value of > 1.8 indicates an **under packed** column and the column should be repacked using an increased flow and/or pressure.