Fast LC

The use of short columns (15-50mm) packed with small particles (i.e., 3 pm) has several significant benefits in the analysis of simple mixtures and in high-throughput screening,18 20 such as  

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High flow-rates (up to 2mLmin-1) fast LC-MS for short columns... 

With recent instrumental development, such as fast LC, fast GC and two-dimensional gas chromatography (GCxGC) and advanced tandem hybrid MS detection systems (i.e., QqTOF, QqLIT, Orbitrap) the analysis of complex mixtures... 

Separation is only as good as the detection of the separated compounds. It is nowadays possible to achieve better separation of compounds than many detectors employed can measure. This means that a close examination of the detection device is required to truly optimize LC/MS. To check the utility of a detector for inclusion in a fast LC system, the following points may help ... 

What is the signal-to-noise ratio under fast LC conditions ... 

When a fast LC system is connected to a detector, care must be taken to ensure that the detector is well suited for the expected flow ranges and peak widths. Most manufacturers, especially those offering dedicated systems for sub-2-micron particle columns, offer efficient UV detectors. Flow rate is usually not an issue for UV and other flow-through cell-based detection systems. However, flow rate can become limiting for dead-end detectors that alter the column effluent, mainly by eliminating mobile phases such as ELSD, CAD, CLND, and mass spectrometers. 

The throughput of fast LC analyses may be significantly improved if cycle times are optimized. The cycle time involves different instrument-dependent and -independent parts. Optimizing the equilibration time and data processing may produce the greatest influence but small additions (seconds) to the cycle time should not be overlooked because optimization of cycle time can increase throughput significantly. 

For non-simultaneously operating multi-sources it is necessary to check whether the data quality achieved is sufficient, especially for fast LC systems with very narrow peaks. A switching source splits the available acquisition time for the offered ionization types and therefore reduces the true acquisition rate of the mass spectrometer. However, simultaneously operating sources do not explicitly show which compound ionized with what technique. If this information is really necessary, two separate runs must be performed. 

Such columns are excellent filters and require more sample preparation to ensure the removal of all solids. To benefit from the full power of LC optimization, the detectors must be optimized as well. Data rates and duty times must match the narrower peaks in very fast (and well resolving) separations. Careful consideration and optimization of all instrument components and software can produce significant cycle time improvements of fast LC separations and further increase throughput. An important aspect of cycle time improvement is parallelization of components of individual analyses. 

MS has recently been used to measure compounds with significant levels of impurities and solubilities below the quantitation limits of other methods. Guo et al.46 described the use of LC/MS for solubility measurements in buffer solutions in a 96-well plate. Fligge et al.47 discussed an automated high-throughput method for classification of compound solubility. They integrated a Tecan robotic system for sample preparation in 384-well plates and fast LC/MS for concentration measurement. This approach is limited by LC/MS throughput. 

Several important aspects relevant to the implementation of fast LC technologies in pharmaceutical laboratories should be mentioned. First, increases in speed should not compromise the quality of the analytical data or the robustness of the chromatography. All methods must be reproducible and validatable to meet the applicable GMP and GLP requirements. Instrumentation should be easily maintained and have minimal downtime. 

To minimize unacceptable interruptions in highly regulated work flows, the smooth transfer of legacy methods from conventional to fast LC methods (via geometric transfer or method redevelopment) is a critical issue for implementing fast LC for pharmaceutical applications. Method transfer from HPLC to UPLC is discussed in detail in the literature.52,53 Moreover, method transfer software that provides parameter conversion between UHPLC and conventional HPLC is available from instrument vendors. 

An overview of HPLC instrumentation, operating principles, and recent advances or trends that are pertinent to pharmaceutical analysis is provided in Chapter 3 for the novice and the more experienced analyst. Modern liquid chromatographs have excellent performance and reliability because of the decades of refinements driven by technical advances and competition between manufacturers in a two billion-dollar-plus equipment market. References to HPLC textbooks, reference books, review articles, and training software have been provided in this chapter. Rather than summarizing the current literature, the goal is to provide the reader with a concise overview of HPLC instrumentation, operating principles, and recent advances or trends that lead to better analytical performance. Two often-neglected system parameters—dwell volume and instrumental bandwidth—are discussed in more detail because of their impact on fast LC and small-bore LC applications. 

For these reasons, smaller-particle columns are particularly well suited for fast LC and high-throughput screening applications. 

With the trend toward using Fast LC and small-bore columns (3-and 2-mm i.d.) in pharmaceutical analysis, increasing demand is placed on reducing the system dispersion or IBW. Figure 15 shows the deleterious effect of IBW on the performance of a Fast LC column showing that... 

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