The attached spreadsheet comprises raw data related to developing an HPLC analytical method, which is used for the analysis of food composition and compounds involved in biomass reactions . The dataset in addition to the incorporated mathematical formulas guide researchers and quality control employees working at food and biomass industries on how to design an experimental design to yield a robust analytical chromatography protocol. Importantly, mathematical formulas furnished with the datasheet feature a user-friendly template for all calculations needed for preparing analytical standards, mix standards, real samples, and the corresponding calibrations. This spreadsheet is composed of two sheets where the first one entitled 'HPLC Standards' deals with the calculations of the concentrations of analytical standards and their relevant calibrations plots. Whereas the second sheet entitled 'Samples Run' is designated for calculating the final concentrations of the samples under analysis. Moreover, the latter provides a concise approach on how to run an HPLC sequence for the quantification of the samples' ingredients. In addition, statistics as well as relative response factor of the used internal standard calculations are demonstrated.
Steps to reproduce
The attached raw data were collected by weighing different amounts of standards used for developing an HPLC method and by DAD output channel (SL DE73458282) set at 210nm wavelength. The chromatographic separation took place using Thermofisher Acclaim organic acids OA 5um 120A (4.0x250mm) column and a mobile phase 200mM Na2SO4 with 0.55ml methanosulfonic acid (pH=2.67). Users are highly recommended to start with preparing individual standards stocks by weighing three different amounts and then fill in the values in the designated cells under each standard name title. The corresponding concentration is based on the density of the used original standard. Therefore, the user is urged to amend the density value according to the used stock chemical standard (right side of the excel sheet). Afterwards, HPLC was run at two or more flow rates depending on the nature of the chosen ingredient and the resulted peak areas are filled in the appropriate cells. As a result, calibration curves are automatically produced. Should the user be interested in making up standard mix solutions of three different concentrations from the original standards stocks, examples of the quantitates transferred are pasted on the right side of the excel sheet under S1,S2, S3, S4, S5, S6. The user has to fill in the transferred amount where the built in formulas calculate the final concentration of each standard in the mix solution. In addition, the calibration curves of the calculated concentrations versus peak areas are plotted down. In regard with running real samples, the second sheet 'Samples Run' is used to fill in the HPLC output peak area values of the standard mix solutions and the samples duplicates. The weight of the prepared sample (m sample) value is equal to the value input under 'Samples Recipes' in the 'HPLC Standards' sheet. Once the peak areas are filled in the appropriate cells, the response factors are calculated automatically. The unknown concentration of the sample ingredient e.g. acetic acid is calculated based on the response factor of the standard mix having the closest peak area to the sample.