Adsorption resin chromatography obtains wide application in characterizing the distribution of hydrophilic/hydrophobic fractions in natural organic matters. As a basic parameter of chromatography, the critical retention factor has a potential impact on results of adsorption and separation. In this study, water samples from a reservoir were fractionated into hydrophilic substances (HIS), hydrophobic acids (HOA), hydrophobic bases (HOB) and hydrophobic neutrals (HON) under the conditions of various critical retention factors (k′cr=5, 10, 25, 50, 100). The distribution of the hydrophilic/hydrophobic fractions were systematically characterized, with special attention placed to their spectroscopic properties. The results showed that the relative proportions of hydrophilic/hydrophobic fractions depended on the value of k′cr, and the proportion of hydrophobic components and the degree of hydrophobicity increased with the increase of k′cr value. In wavelength range of 250~280 nm, the UV absorbance of HIS increased with increasing k′cr, while the absorbance of hydrophobic fractions performed in the reserved trend. The difference in UV spectra between the hydrophilic and hydrophobic fractions was enlarged with increasing k′cr value. The specific UV absorbance (i. e. UV absorbance per unit concentration) of the hydrophobic fractions was found to be sensitive to the value of k′cr, indicating that the critical retention factor may affect the properties of aromatic functional groups in the obtained fractions. Three dimensional excitation-emission matrix fluorescence spectroscopy was further conducted to characterize the hydrophilic/hydrophobic fractions, with the spectral data analyzed using fluorescence regional integration and fluorescence index techniques. The results revealed that the fluorescence peak appearance (peak location and intensity), fluorescence regional distribution and fluorophore density were related to the value of k′cr, meanwhile the fluorescence indices of BIX, HIXem and Peak T/C were sensitive to the value of k′cr. It is speculated that the critical retention factor may have profound impact on the chemical compositions and transformation behaviors of the resultant hydrophilic/hydrophobic fractions. Therefore, it is suggested that particular attention should be paid to the setting of the critical retention factor and indicating its value clearly when conducting separating and characterizing of the distribution of hydrophilic/hydrophobic fractions.