Automation HPLC sampling, optimization

The development of a chromatographic procedure for an unknown sample (mixture) requires the selection of a variety of experimental conditions (type and composition of the mobile phase, characteristics of the column and the stationary phase, temperature, flow-rate, pressure, type of gradient, etc.). This problem was traditionally solved in an empirical way and with the aid of the vast literature on similar situations already dealt with. The last few years have seen attempts at the rationalization and automation of the optimization of chromatographic processes which have resulted In Interesting systematic approaches of great use. The monographs by Berridge [56], devoted to HPLC, and that by Shoenmakers [57], which deals with both HPLC and GC, represent the most systematic and complete compilations in this field at present. 

Sample preparation, injection, calibration, and data collection, must be automated for process analysis. Methods used for flow injection analysis (FLA) are also useful for reliable sampling for process LC systems.1 Dynamic dilution is a technique that is used extensively in FIA.13 In this technique, sample from a loop or slot of a valve is diluted as it is transferred to a HPLC injection valve for analysis. As the diluted sample plug passes through the HPLC valve it is switched and the sample is injected onto the HPLC column for separation. The sample transfer time typically is determined with a refractive index detector and valve switching, which can be controlled by an integrator or computer. The transfer time is very reproducible. Calibration is typically done by external standardization using normalization by response factor. Internal standardization has also been used. To detect upsets or for process optimization, absolute numbers are not always needed. An alternative to... 

HPLC is extremely useful in monitoring and optimizing industrial processes. Conventional process monitors measure only bulk properties, such as the temperature and pressure of a reactor, while HPLC permits continuous realtime monitoring of consumption of starting materials, product composition, and impurity profile. There are a number of new initiatives relevant to HPLC for process monitoring, including sample preparation, automation, miniaturization, and specialized detectors. 

Mobile phase optimization using HPLC can easily be carried out using fully automated instruments, but this approach is extremely time-consuming. Conversely, modern thin-layer chromatographic techniques allow several mobile phases to be tested in parallel within a very short period of time. Furthermore, different methods are available to visualize the sample components, and the thin-layer chromatogram immediately provides information about the presence of products in the sample that remain at the point of application. 

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