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Introduction
Preparative HPLC and LC processes will isolate and purify milligram to kilogram amounts of compound. The technique uses larger particle size silicas, polymers and wider internal diameter columns than in analytical HPLC. Column efficiency can be preserved on scale-up from analytical to preparative separations. However, broader lower efficiency chromatographic peaks are more often observed when the column is used in an overload state.
Separation Criteria
The criteria governing preparative separations are very similar to those influencing analytical HPLC. Economic considerations become more important. They are governed by four factors.
• Resolution
Separation Strategy: By optimising the separation between the peak of interest and the nearest contaminant, high sample loads can be achieved without compromising product purity.
• Loadability
Loadability is controlled by the silica particles’ pore size and available surface area. The smaller the pore size the larger the surface area. The comparative loadability of different pore size silicas is shown in Figure 1 below. Application of the smaller pore size silicas is limited by the range of molecular weight materials they can chromatograph.
• Chemical Stability
The lifetime of a column is often dependent on the silica’s chemical stability. Conditions of use will be very important.
• Physical Stability
The robustness of a silica will determine how many times a material can be repacked into a column. Reversed-phase is the dominant technique used in analytical HPLC. Normal-phase HPLC is still often used in preparative separations due to the high cost of reversed-phase materials and the easier recovery of solute from the organic solvents used.
Two strategies dominate the approach to preparative HPLC. In the ‘scale-up’ approach a method developed for analytical purposes is directly applied to a larger i.d. column. Although typical 3 - 5µm particles may be replaced with 10µm material of identical selectivity, high preparative efficiencies are maintained. Such an approach is particularly suitable for purifying gram quantities of material with low k'-values.
In the alternative ‘overload’ approach resolution is sacrificed by operating the column in an overload situation. Such high loadings maximise column capacity. Separations are poorer but gram to kilogram amounts of material may be purified.
A comparison of scale-up parameters is highlighted in Table 1.
Table 1. Column Capacity
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