HPLC operation summary

The following is a summary of important procedures or sequence of events for HPLC operation ... 

A summary update of best practices in HPLC operation, method development, maintenance, troubleshooting, and regulatory compliance. 

Fully automated analysis is also an option wherein the samples are placed in an autosampler and predefined HPLC-NMR experiments are performed. This is covered in detail elsewhere in this volume, but in summary, the software allows automatic detection of UV peaks in the chromatogram based on predetermined time-windows or peak intensities. The successful detection of each UV peak triggers the system to stop the flow at an appropriate time to isolate the peak in the NMR flow probe. Then data relating to the peak (intensity, retention time, etc.) are transferred to the NMR host computer and used to define the parameters for the automatically acquired NMR spectrum. This automatic NMR operation includes field homogeneity optimisation, setting and optimisation of all NMR acquisition parameters, and the predefinition of the resultant signal-to-noise ratio required in the spectrum. The measurement of 2-dimensional (2D) NMR spectra can also be performed. 

In summary, more effective and successful HPLC assays can be accomplished by following some of the best practices in mobile phase preparation, strategies in qualitative, quantitative, and trace analysis, and some of the standard operating procedures in HPLC system and data system operation. 

System calibration refers to the periodic operational qualification of the HPLC, typically every 6 to 12 months in most regulated laboratories. This calibration procedure is usually coordinated with an annual preventative maintenance (PM) program and is performed immediately after PM. A calibration sticker is placed on the instrument to indicate its calibration status and readiness for GMP work. The reader is referred to the principles and strategies behind HPLC calibration criteria published elsewhere.8 A summary of the calibration procedures and acceptance criteria, including additional procedures recommended for initial operational qualification, is listed in Table 9.3. 

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