Preparative HPLC optimization

Preparative HPLC optimization goals which ultimately lead to a product with a given minimum purity may include the maximum amount of the purified material per weight unit of stationary phase per time unit (g/kg/day), the maximum amount of the purified material per mobile phase unit per time unit (g/L/day), the maximum production rate (g/day), the lowest cost ( /kg), the maximum recovery (%), and the maximum production rate with maximum recovery. Regardless of the differences in application, it is important to be aware of the following parameters that may affect the purity and recovery of the product as well as the time and cost required for the separation ... 

The aim of this work, to develop a RP-HPLC method for the detemtination of four components given above in a new symp preparation by optimizing the experimental conditions using two level fractional factorial design. 

Taylor and co-workers further demonstrated the value of open-access LC/MS systems for generating a widened scope of pharmaceutical analysis applications, including (1) characterization of synthetic intermediates and target compounds (2) reaction monitoring (3) reaction optimization (4) analysis of preparative HPLC fractions and (5) analysis of thin layer chromatography (TLC) plate spots. The availability of these methods led to the increased use of LC/MS for structural analysis. The short analysis time and reliable structure confirmation resulted in the use of LC/MS as a first choice for structure characterization for synthetic chemistry applications, as well as an expanded, and perhaps, integrated role of sample generator and analyst. 

Clearly it is not possible to describe every technique, or variation in a particular mode of chromatography, so this chapter will concentrate on the most dominant approaches to optimization of preparative HPLC. The objective here is to maximize the column load and to minimize mobile phase wastage allowing the purification of the largest amount of target in the minimum space. 

Analytical chromatographic options, based on linear and nonlinear elution optimization approaches, have a number of features in common with the preparative methods of biopolymer purification. In particular, both analytical and preparative HPLC methods involve an interplay of secondary equilibrium and within the time scale of the separation nonequilibrium processes. The consequences of this plural behavior are that retention and band-broadening phenomena rarely (if ever) exhibit ideal linear elution behavior over a wide range of experimental conditions. First-order dependencies, as predicted from chromatographic theory based on near-equilibrium assumptions with low molecular weight compounds, are observed only within a relatively narrow range of conditions for polypeptides and proteins. 

The success of a particular analytical or preparative HPLC strategy with polypeptides or proteins is predicated by the ease of resolving to a predefined level the desired component from other substances, many of which may exhibit similar separation selectivities but are usually present at different abundance levels. For high-resolution purification procedures to be carried out efficiently, it is self-evident that rapid, multistage, high-recovery methods must be utilized. To minimize losses and improve productivity, on-line, real-time evaluation of each of the recovery stages is an essential objective. Furthermore, overall optimization and automation of the individual unit operations must be achieved. Similar criteria but with different endpoints apply in high-resolution analytical application. 

Not all of the separation parameters can be improved simultaneously and the optimization process often requires multiple experiments. In addition, separation problems, such as the sample bleeding through the column during loading or the product not completely eluting from the column, should be identified and corrected. Steps of the HPLC optimization experiment include (a) preliminary recording of the peak area-concentration plot for a pure product standard, (b) calculation of the amount of the target material in the solution prepared for separation, (c)... 

Preparative HPLC separations are used in all stages of process development. The three main stages in process development as well as examples for the tasks and corresponding criteria for process development are described in Fig. 4.1. They differ in time pressure, frequency of the separations and amount of target products, which dominate the economic structure of the process. In laboratory and technical scale the development of the separation is dominated by the time pressure and the separation problem includes only a limited amount of sample and a limited number of repetitions. Process optimization in this small- to semi-scale is not helpful, because the effort for optimization will not be repaid in reduced operational costs. 

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