One of the beauties of purification using preparative HPLC is the simplicity of how scaling of a separation process is made. This article will give you an insight into how this is done, and which difficulties may occur.
Since the chromatographic process is easily scalable, all method development can be performed using analytical sized columns. The columns should be packed with material suitable for preparative purification (i.e. silica particles ranging from 7 to 25 µm). As long as the stationary phase type and silica brand is the same, one can be sure that the properties of the stationary phase will be the same regardless of batch size.
The method development work normally performed on a 4.6 x 250 mm column will provide the most important parameters needed. Together with target criteria such as product purity, acceptable yield loss, production demand and pressure limitations one has all the information needed for a successful process scaling.
By keeping the bed length and particle size constant, scaling is performed by calculating the cross-sectional area ratio between the analytical column and the column intended to be used for production. The ratio is the scaling factor and will be used to calculate other key parameters such as loading, flow rate and eluent consumption.
|Internal diameter (I.D.) [cm]||0.46||10||30||80|
|Cross section area [cm²]||0.17||79||707||5030|
For example, method development has shown that a certain peptide is suitable to purify using RP-HPLC. The purity demand is 99% while 20% crude loss is acceptable. Furthermore, a 1% column loading [g crude/kg resin] is possible and the mobile phase is a buffer/MeOH gradient. Pressure limitation of the production scale system is 70 bar enabling the use of 10 µm silica resin and a linear flow rate of 6 cm/min. Below are tables summarizing the input data and showing key process data:
If the scaling work has been performed properly and problems, such as decreased chromatographic resolution, still occur there are a few things important to have in mind.
The first issue is the decreased bed stability due to removal of the wall effect that helps to stabilize beds packed in analytical columns. This negative effect is greatly reduced by applying a constant piston pressure on the bed which removes any formation of bed void volumes which would be detrimental to the chromatographic performance. Today, column manufacturers have sophisticated column designs ensuring that this problem is kept to a minimum. In fact, there are examples of columns with 1.6 m diameter!
Another problem is in various ways related to axial and radial temperature gradients forming in the column. This will in different ways distort the symmetry of the peaks. To reduce this problem one should always premix the mobile phase so that mixing enthalpy will not affect the separation.
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