Key Considerations: Part 2

Figure 3. Influence of Temperature on Preparative Chromatography Performance

Most preparative chromatography is not performed in a column oven due to the size of the column which would struggle to fit into conventional column ovens. Stationary phases can be stored in Column Hubs, but these are not temperature controlled like a conventional HPLC oven. As such, it is preferable to evaluate the column temperature at room temperature.  

One of the major factors for working at room temperature relate to the column dimensions. As the column diameter increases, the temperature gradient within the column becomes more pronounced. This causes the peaks to become broader due to the parabolic flow of the analyte band migrating through the column. The band moves faster at the wall of the column, than the middle of the column, which causes the band to disperse, and reduce the efficiency of the fraction. This causes detrimental peak shapes as illustrated in Figure 3.   

Figure 4. Changes in Selectivity Following Organic Solvent Substitution from Acetonitrile to Methanol

Table 1. Common HPLC Buffer Systems, pH Ranges and Volatility Characteristics

This is investigated by varying the injection amount in stages using the same concentration sample. When increasing the injection volume, it is necessary to pay special attention to the effect of the sample solvent.  

It is necessary to pay special attention to the effect of the sample solvent. In cases where the sample solvent has a stronger elution power than the mobile phase, the peak shape may deteriorate depending on the injection volume (a tendency to exhibit leading – the red trace in the chromatogram in Figure 4). For example, in reversed phase mode, a high proportion of methanol can often lead to detrimental peak shape. When the sample diluent is weaker than the elution strength of the mobile phase, the peak is not adversely affected by broadening. 

Since it greatly depends on the column internal diameter, it is important to pay attention to the injection volume when reducing the column ID.

Set the target pH based on that information when the structure and pKa of the compound are known. The buffer should be selected so that the buffering capacity is near the set pH. It is important to choose volatile salts or acids in cases where concentration or drying is anticipated, or if mass spectrometry is used as the mode of detection.  

Figure 5. Analytical Method Development Support Software LabSolutions MD

Method development is a difficult activity for most separations. Shimadzu’s LabSolutions MD can assist with this process, by providing a platform for screening, method development and robustness testing on an analytical scale which can then be translated to the preparative scale downstream. Criteria can be set for the automatic exploration of gradient conditions which allows for seamless method development for tricky compounds. 

Figure 6. Automated Method Scale-Up from Analytical to Preparative HPLC

Figure 7. Automated Method Transfer from Analytical to Preparative HPLC Systems

Moving from the analytical scale to the preparative scale can be tricky if you don’t remember all the parameters which need to be adjusted for the different column formats. This includes: 

• Injection volume
• Flow rate
• Gradient conditions

LabSolutions MD can convert an analytical scale method to prep scale just by adding the column information and flow rate. This ensures the method can be simply scales with fewer technical issues.