Technical Information

Overview of method development for isolation/purification Analytical scale conditions Loading Scale-up Preparative separation conditions Effective preparative separation technique Examples of preparative purification


1. Method development on the analytical scale

To develop preparative purification conditions, the first stage is to study the separation on the analytical scale. Columns should be selected taking into account availability of preparative columns/packing materials and costs for later scale up to the preparative separation. The post-column sample treatment should also be taken into account when selecting the separation conditions.

Separation mode (Packing material)

The following points should be considered for the selection of an appropriate separation mode.

  1. Separation efficiency: Resolution, Loadability, Run time
  2. Cost performance: Costs (packing materials/mobile phases), Throughput
  3. Others: Safety, Characteristics of the target substances, Specification of the preparative LC system

Separation method

For the preparative separation, it is important to separate the target compounds efficiently with high purity and high recovery rate.


Mobile phase

It is recommended to use analytical-scale columns for mobile phase screening. Consideration of loading amount and efficiency on the post-column sample treatment should also be taken into account for preparative separation.
With isocratic elution, recycling preparation is possible. In addition, there is no need for reequilibration between sample injections during repeat preparative separations. With gradient elution, preparative fraction volumes can be expected to be smaller because peak widths will be narrower than those for an isocratic elution.

Comparison between isocratic elution and gradient elution

Column YMC-Pack C4 (5 µm, 300 Å) 250 X 4.6 mmI.D.
Eluent A) water/TFA (100/0.1)
B) acetonitrile/TFA
Temperature 40ºC
Detection UV at 214 nm

Post-column treatment is easier if volatile additives are used to control mobile phase pH


Generally, column temperature is controlled with a column oven for analytical separation, therefore, it is preferable for preparative separations to be performed under room temperature. It is not recommended to conduct preparative separations under other than room temperature because it is difficult to keep the internal column temperature constant across the column with a large inner diameter even with a large-scale temperature controlling device. Non-uniform internal temperatures lead to poor peak shape or peak splitting due to uneven flow of the sample. Therefore, it is important to check the temperature dependence of the resolution and peak shape on an analytical scale and to choose separation conditions at room temperature wherever possible for efficient preparative separations.