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Size exclusion chromatography with total organic carbon detection (HPSEC-TOC) is a widely employed technique for characterizing aquatic natural organic matter (NOM) into high, medium, and low molecular weight fractions. This study validates the suitability of HPSEC-TOC for a simplified yet efficient routine analysis of freshwater and its application within drinking water treatment plants. The investigation highlights key procedural considerations for optimal results and shows the importance of sample preservation by refrigeration with a maximum storage duration of two weeks. Prior to analysis, the removal of inorganic carbon is essential, which is achieved without altering the NOM composition through sample acidification to pH 6 and subsequent N2-purging. The chromatographic separation employs a preparative TSK HW-50S column to achieve a limit of detection of 19.0 µgC dm−3 with an injection volume of 1350 mm−3. The method demonstrates linearity up to 10,000 µgC dm−3. Precision, trueness and recovery assessments are conducted using certified reference materials, model compounds, and real water samples. The relative measurement uncertainty in routine analysis ranges from 3.22% to 5.17%, while the measurement uncertainty on the bias is 8.73%. Overall, the HPSEC-TOC represents a reliable tool for NOM fractions analysis in both treated and untreated ground and surface water. [ABSTRACT FROM AUTHOR]