Economical Preparative HPLC (Part 1 of 2)
Criteria for an efficient and economical preparative HPLC
Preparative HPLC is one of the most important methods for the efficient clean-up of chemical and biological substances from synthesis or extraction mixtures. It is used for separation of few milligrams of valuable end or intermediate products on the laboratory scale up to gram and kilogram amounts in pilot plants or production facilities. Often, similar purities cannot be achieved with other clean-up methods, like e.g. recrystallization.
Several criteria are important for selection of suitable columns or adsorbents in up-scaling from analytical to economical preparative HPLC.
Requirements for preparative separations
The target of analytical HPLC is a separation as complete as possible of individual components of a mixture with subsequent quantification of the analytes.
The goal of a preparative separation is the purification (isolation) of a desired product (or set of products) with acceptable purity and yield of final product defined by an economical mode of operation. Thus, the requirements of a preparative separation – in terms of purity and yield of the products, as well as sample throughput – must be considered for the selection of a suitable preparative HPLC system.
High purity of the product can be achieved by high resolution between the product and the neighboring peaks. The resolution of two peaks is determined by the chromatographic system of mobile and stationary phase. A smaller particle size of the stationary phase (e.g. 5 or 10 µm) combined with the adjustment of further system parameters facilitates higher resolution and thus higher purity.
Yield is also important for preparative clean-up. This is particularly important for multi-step syntheses. Each step should have a high yield as possible else the final overall yield can be very low. The yield can be increased by use of columns with larger inner diameters. Established sizes for semi-preparative scale are inner diameters from 8 to 21 mm ID and from 21 to 80 mm ID on the preparative scale.
Alternatively, for smaller inner diameters the yield can be increased by higher throughput of samples (higher number of separation runs).
Finally, an individual economic decision between the maximization of purity, yield and throughput is necessary for the introduction of a preparative HPLC method.
Scale-up from analytical separation to preparative scale
Prior to development of a preparative separation method, an analytical method with a sufficient separation of main and side components should be established. The selection of the analytical column with regard to an efficient phase (selectivity, pH and pressure stability, loadability, availability of suitable particle sizes) should be made with the prospect of a scale-up to the preparative scale. The chromatographic conditions should be selected such that the sample mixture is separated with a good resolution, preferably under isocratic conditions. As a general rule, gradients are not recommended for preparative separations, because they always imply a great effort and with it higher operating costs. Good resolution for the analytical chromatogram offers the advantage, that the load on the preparative column can be higher. Hence, the yield can be increased.
For scale-up to the preparative scale a column with the same length but with a larger inner diameter is selected. In reference to the scale-up formula the loading amount of the sample is proportional to the cross-section of the column.
The eluent flow has to be adapted to the larger inner diameter to achieve similar retention times by comparison with the analytical column.
Due to overload effects the amount of sample loadable on the column is considerably increased; a simple up-scaling would be uneconomical with respect to eluent and adsorbent needs. One distinguishes concentration, volume and mass overload.
For concentration overload the concentration of the sample substances is increased while the injection volume remains constant. With increasing overload a gradually growing peak deformation results, which approximates the shape of a triangle. A concentration overload can only be realised, if the sample substances have sufficient solubility in the sample solution. Often DMSO is used as injection solvent, which requires a high stability of the column.
For low solubility of sample substances an overload can only be achieved by continuously increasing the injection volume with the same sample concentration.
The peaks approach the shape of a rectangle with this volume overload.
The term mass overload is used, if the injected sample mass (calculated from injection volume and concentration) exceeds a certain value, at which the local concentration of the sample in the column is so large, that an equilibration is no longer possible. The mass overload gives unintentional changing of the retention volumes of the peak fronts and peak broadening with tailing.
To get as much pure product per time unit as possible, the column is almost always operated in an overloaded status. A combination of concentration and volume overload is most often used in practice. Volume overload rather results for diluted sample solutions while concentration overload is observed for concentrated samples. In many cases both effects exist and the peaks become trapezoidal. For strongly concentrated samples, additionally mass overload may appear, which for columns with low loadability results in premature tailing. In general, the target is approximation to the case of concentration overload, because a greater sample amount can be separated.
The displayed up-scaling table shows typical sample amounts in relation to RP phases. Whereas 0.02 – 2 mg sample can be loaded onto an analytical column with 4 mm ID, the amount increases to 0.6 – 60 mg for a preparative column with 21 mm ID with the linear scale-up factor of 27.6, and to 3 – 350 mg for 50 mm ID with factor 161.3.
Of course the stated maximal amounts always depend on the separation problem, the sample composition and the quality of the preparative column in relation to loadability, selectivity and base deactivation. The mentioned values can be under-run as well as considerably exceeded.