The binding between four matrices (beaded cellulose, cellulose acetate, cellulose triacetate and Sepharose CL‐6B) and beaded cellulose derivatized with a thiacarbocyanine dye with 5′‐mononucleotides is investigated by Saturation Transfer Difference Nuclear Magnetic Resonance (STD–NMR) technique. This procedure intends to identify unspecific interactions between 5′‐mononucleotides and matrices commonly used in affinity chromatography systems and also clarify the contribution of a thiacarbocyanine dye immobilized onto cellulose beads in a biorecognition process. The differences between non‐derivatized and derivatized beaded cellulose evidence the contribution of thiacarbocyanine dye in the observed interaction. STD–NMR experiments show that Sepharose CL‐ 6B interact less with the 5′‐mononucleotides comparatively with beaded cellulose. Indeed, beaded cellulose shows nonspecific interactions with almost all 5′‐mononucleotides that compromises the specificity of the interaction between the thiacarbocyanine dye immobilized with the 5′‐mononucleotides. The cellulose matrices where the hydroxyl groups are replaced by acetate and triacetate groups do not exhibit binding response to the 5′‐ mononucleotides, whereas the thiacarbocyanine dye contribution is evidenced by the reinforcement of the interactions with the sugar moiety of 5′‐GMP and 5′‐UMP and with base of 5′‐AMP, 5′‐CMP and 5′‐TMP. This screening of the nucleotide atoms involved in the binding to the supports can be very useful in chromatography evaluations in which dye‐affinity chromatography supports may be used, such as purification of nucleic acids. Copyright © 2011 John Wiley & Sons, Ltd.