HPLC method development information

Formore information, see L. R. Snyder, j. J. Kirkland, and J. L. Glajch. Practical HPLC Method Development. 2nd ed.. New York Wiley. 1997... 

In the development of a SE-HPLC method the variables that may be manipulated and optimized are the column (matrix type, particle and pore size, and physical dimension), buffer system (type and ionic strength), pH, and solubility additives (e.g., organic solvents, detergents). Once a column and mobile phase system have been selected the system parameters of protein load (amount of material and volume) and flow rate should also be optimized. A beneficial approach to the development of a SE-HPLC method is to optimize the multiple variables by the use of statistical experimental design. Also, information about the physical and chemical properties such as pH or ionic strength, solubility, and especially conditions that promote aggregation can be applied to the development of a SE-HPLC assay. Typical problems encountered during the development of a SE-HPLC assay are protein insolubility and column stationary phase... 

Chovan et al.30 described a system that integrates different components of bioanalysis including automatic in vitro incubation, automatic method development (mainly SRM transitions for LC/MS/ MS analysis), and a generic LC method for sample analysis to minimize human intervention and streamline information flow. Automaton software (Applied Biosystems) was used for automatic MS method development. Flow injection was used instead of a HPLC column to decrease run time to 0.8 min per injection. Two injections were performed. The first was performed to locate the precursor ion and optimal declustering potential (DP). The second injection was performed to locate the product ion and optimal collision energy (CE). 

The goal of ECAT is to provide assistance to the user of a chromatograph in the development of an HPLC method. To do this, one must specify the tasks performed in developing an analytical method. The computer performs these tasks by processing information. In ECAT we are calling the collection of information specific to a task a Module. The modules and information flow which will be needed for the completely implemented ECAT are shown in Figure 2. 

Many researchers have put a considerable amount of effort into studies of the chiral recognition mechanisms (using, e.g., NMR and molecular modeling), but yet the choice of chiral selector or chiral phase for a new compound is often based on trial and error. Different strategies for chiral method development have been presented by many of the retailers of chiral columns as a service for the customers. In addition to the information supplied by these retailers, another source of knowledge is Chirbase, a database that contains more than 50,000 HPLC separations of more than 15,000 different chiral substances [61], which also can provide guidance to the analytical chemist. 

Minimal sample preparation (dilution in HPLC mobile phase) is necessary. A standard reversed-phase HPLC method is used for all the samples associated with a drug candidate to reduce time-consuming method development/method refinement procedures. Standard reversed-phase methods typically involve a 20-30 min cycle time and provide information on a wide range of compounds. The incorporation of a standard method strategy allows the use of autosampling procedures and standard system software for data analysis. 

Steuer et al. compared supercritical fluid chromatography with capillary zone electrophoresis (CZE) and high-performance liquid chromatography (HPLC) for its application in pharmaceutical analysis [24]. Efficiency, performance, sensitivity, optimization, sample preparation, ease of method development, technical capabilities, and orthogonality of the information were the parameters studied. They concluded that SFC is ideal for moderately polar compounds, such as excipients, for which mass detection is required. 

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