wide range of peak base widths and retention times exhibited by the twenty analytes across the tested conditions. In order to reduce this complex dataset to a more tractable form, it was necessary to identify criteria that would simplify evaluation and comparison of the separation quality. As high-throughput untargeted LC- or SFC-MS metabolic phenotyping studies require high peak capacities and resolution within a short analysis time, the quality of separation for each combination of column, modifier and temperature was evaluated based on three parameters: peak base width, sum of resolution and the normalised product of the resolution. The peak base width may be related to peak capacity as follows: (1) where Pc is the peak capacity for a gradient of length tg, and wp is the base width for each of n peaks used in the calculation [30]. Another common definition of peak capacity uses the sum of the resolution () across the chromatogram: (2) where RS is the resolution between peaks, calculated here using equation 3: (3) where wp,n is the peak width for the nth peak, eluting at retention time tr,n. Both definitions for peak capacity have constraints on their use to describe gradient separations (e.g. equation 2 applies to situations where peak widths follow a similar pattern across the chromatogram [30]), but are sufficient for our purpose viz. comparison of separation quality under different chromatographic conditions. Thus, based on equations 1 and 2, the average peak base width and the average may be used to compare the relative peak capacities of the evaluated chromatographic conditions. In the current analysis, we have chosen to compare peak base width and resolution data using the boxplot representation, and hence are comparing the median rather than the average parameters across conditions; however, where relevant, we have also provided mean values within the text. One shortcoming of peak capacity as an estimate of chromatographic performance is that it does not take into account the distribution of peaks across the chromatographic window. While minimising peak base width and increasing resolution are crucial for improved separation in metabolic profiling studies, an even distribution of peaks across the retention time range ensures a more efficient use of chromatographic space. To evaluate peak distribution, Schoenmakers and others have used the normalised resolution product ( or NPR) [31]:
wide range of peak base widths and retention times exhibited by the twenty analytes across the tested conditions. In order to reduce this complex dataset to a more tractable form, it was necessary to identify criteria that would simplify evaluation and comparison of the separation quality. As high-throughput untargeted LC- or SFC-MS metabolic phenotyping studies require high peak capacities and resolution within a short analysis time, the quality of separation for each combination of column, modifier and temperature was evaluated based on three parameters: peak base width, sum of resolution and the normalised product of the resolution. The peak base width may be related to peak capacity as follows: (1) where Pc is the peak capacity for a gradient of length tg, and wp is the base width for each of n peaks used in the calculation [30]. Another common definition of peak capacity uses the sum of the resolution () across the chromatogram: (2) where RS is the resolution between peaks, calculated here using equation 3: (3) where wp,n is the peak width for the nth peak, eluting at retention time tr,n. Both definitions for peak capacity have constraints on their use to describe gradient separations (e.g. equation 2 applies to situations where peak widths follow a similar pattern across the chromatogram [30]), but are sufficient for our purpose viz. comparison of separation quality under different chromatographic conditions. Thus, based on equations 1 and 2, the average peak base width and the average may be used to compare the relative peak capacities of the evaluated chromatographic conditions. In the current analysis, we have chosen to compare peak base width and resolution data using the boxplot representation, and hence are comparing the median rather than the average parameters across conditions; however, where relevant, we have also provided mean values within the text. One shortcoming of peak capacity as an estimate of chromatographic performance is that it does not take into account the distribution of peaks across the chromatographic window. While minimising peak base width and increasing resolution are crucial for improved separation in metabolic profiling studies, an even distribution of peaks across the retention time range ensures a more efficient use of chromatographic space. To evaluate peak distribution, Schoenmakers and others have used the normalised resolution product ( or NPR) [31]:
Accepted%20160411-JCA-15-2362R1.docx