The physicochemical properties and chemical reactivity of dissolved organic matter (DOM) are of tremendous practical significance in both natural and engineered aquatic and terrestrial systems. DOM is frequently extracted, fractionated, and concentrated from environmental samples using a variety of operationally defined physical and chemical processes in order to facilitate study of specific physicochemical properties and aspects of its chemical reactivity. This study was conducted to systematically examine the influence of operationally defined physical and chemical characterization methods on observed DOM physicochemical properties and reactivity with aqueous chlorine. The effects of chemical separation were evaluated by applying an existing resin adsorption based procedure which simultaneously extracts and fractionates DOM and inorganic constituents into hydrophobic and hydrophilic acid, base, and neutral dissolved material matrix (DMM) fractions. Physical separation based on DOM apparent molecular weight (AMW) was also evaluated using batch ultrafiltration (UF) data in conjunction with a suitable membrane permeation model. Linear independence of membrane solute transport was theoretically described using non-equilibrium thermodynamics and experimentally demonstrated for AMICON® YC/YM series UF membranes. Mass balances on DMM fraction constituents in untreated and previously coagulated natural waters indicated that quantitative recovery (100 ± 2%) of DOM constituents was achieved, while recovery of inorganic constituents such as iron and aluminum was substantially incomplete (30%-74%). Comparison of whole-water DOM properties with those mathematically reconstituted from DMM fractions demonstrated a marked shift in DOM properties toward lower AMW. Evidence of pH induced partial hydrolysis of protein, polysaccharide, and ester DOM components was also observed. Decreased specific Cl2 demand (mmol DCl2/ mmol DOM) and specific trihalomethane formation (mmol THM/mmol DOM) following chemical fractionation were attributed to increased molar DOM concentration and decreased DOM association with colloidal iron oxide surfaces. Collectively, the results of this research indicate that operational characterization methods result in alteration of DOM physicochemical properties and reactivity with aqueous chlorine, and caution is therefore advisable when interpreting the results of studies conducted using chemically extracted or fractionated DOM.