Conditional affinity spectrum underlying NICA isotherm provides practical insight into the characteristics of metal ions binding to HS at constant

environmental conditions. The distributions of binding affinity for bound species are estimated. Carboxylic groups make the main contributions to Cu

binding to HS. For JLHA with relatively high amount of carboxylic groups, the occupation of phenolic type sites by Cu can be negligible. Phthalate-type

structure is more probable for Cu binding to JLHA. There is a non-negligible amount of phenolic-Cu on JGHA, which increases the chance of forming more

stable complexes.Binding of Cu(II) to soil fulvic acid (JGFA), soil humic acids (JGHA, JLHA), and lignite-based humic acid

(PAHA) was investigated through NICA-Donnan modeling and conditional affinity spectrum (CAS). It is

to extend the knowledge of copper binding by soil humic substances (HS) both in respect of enlarging

the database of metal ion binding to HS and obtaining a good insight into Cu binding to the functional

groups of FA and HA by using the NICA-Donnan model to unravel the intrinsic and conditional affinity

spectra. Results showed that Cu binding to HS increased with increasing pH and decreasing ionic

strength. The amount of Cu bound to the HAs was larger than the amount bound to JGFA. Milne’s generic

parameters did not provide satisfactory predictions for the present soil HS samples, while materialspecific

NICA-Donnan model parameters described and predicted Cu binding to the HS well. Both the

‘low’ and ‘high’ concentration fitting procedures indicated a substantial bidentate structure of the Cu

complexes with HS. By means of CAS underlying NICA isotherm, which was scarcely used, the natureof the binding at different solution conditions for a given sample and the differences in binding mode

were illustrated. It was indicated that carboxylic group played an indispensable role in Cu binding to

HS in that the carboxylic CAS had stronger conditional affinity than the phenolic distribution due to its

large degree of proton dissociation. The fact was especially true for JGFA and JLHA which contain much

larger amount of carboxylic groups, and the occupation of phenolic sites by Cu was negligible.

Comparable amounts of carboxylic and phenolic groups on PAHA and JGHA, increased the occupation

of phenolic type sites by Cu. The binding strength of PAHA-Cu and JGHA-Cu was stronger than that of

JGFA-Cu and JLHA-Cu. The presence of phenolic groups increased the chance of forming more stable complexes,

such as the salicylate-Cu or catechol-Cu type structures.