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Polymeric materials with immobilized metal nanoparticles are widely used in various fields of chemistry, physics, and biology, which largely stimulates their further research. Due to their small size, they exhibit unique structural, magnetic, catalytic, biological properties, uncharacteristic for particles of large size. Polymers modified with metal nanoparticles are promising to be used as stationary phases for HPLC because of their high mechanical, chemical and thermal stability. Also, they can be easily chemically modified that allows programming the properties of the polymer matrix. The microspheres of a copolymer styrene-divinylbenzene (CSD) (3.3 μm) with a degree of cross-linking of 50% was used as the carrier of the GNPs. The polymer was modified directly by gold nanoparticles (GNPs), stabilized with citrate ions, 10 nm in size, and then the citrate ions were replaced with L-cysteine. A scanning electron microscope (SEM) was used to evaluate the surfaces of the obtained copolymer. The GNPs were rather evenly adsorbed onto the non-functionalized surface of the sorbent. In addition, the diffuse reflectance (DR) method was applied to measure the spectra of the unmodified CSD, gold-coated CSD and the SiO2-Au-L-cysteine sorbent. These studies showed greater adsorption of gold nanoparticles to the polymer matrix than to the surface of silica gel. It is confirmed by the results of the SSD-Au-L-cysteine sorbent study with atomic-emission spectroscopy and mass spectrometry with inductively coupled plasma. It was shown that 6 wt. % of gold was adsorbed onto the surface of the copolymer. The obtained sorbent was used as a stationary phase for HPLC to separate nitrogen-containing organic compounds and their enantiomers. The chromatographic behavior of the analytes was investigated depending on the mobile phase composition, in particular the nature of the buffer solution, its pH and concentration, and the content of the organic solvents. The effect of acetonitrile concentration was also studied in the range of 80-100%. The decrease of acetonitrile concentration enhances the interaction of analytes with sorbent surface and leads to increase of retention. We showed that novel method for immobilization of L-cysteine on copolymer styrene-divinylbenzene surface via gold nanoparticles. The developed technique is a good alternative to existing approaches and very promising for future investigation and application.