20.500.12556/RUL-184249 Študij kromatografskega obnašanja modelnih proteinov na različnih kationsko-izmenjevalnih monolitnih kolonah Study of chromatographic behavior of model proteins on different cation-exchange monolithic columns Kationsko-izmenjevalna kromatografija je ena ključnih metod za ločevanje in čiščenje proteinov, pri kateri na zadrževanje proteinov poleg osnovnih elektrostatskih interakcij pogosto vplivajo tudi ostale fizikalno-kemijske lastnosti ionsko-izmenjevalne skupine in matrice kromatografske kolone. Namen dela je bil primerjati delovanje treh različnih kationsko-izmenjevalnih monolitnih kolon, CIM SO3, CIM SO4 1 in CIM SO4 2, ki so se razlikovale po tipu izmenjevalne skupine, gostoti liganda in sestavi matrice. Zanimalo nas je, kako navedene razlike vplivajo na mehanizem adsorpcije dveh modelnih proteinov – citokroma c in lizocima. Kromatografsko obnašanje modelnih proteinov smo najprej preučili z elucijo v linearnem solnem gradientu. Ionsko-izmenjevalni proces smo nato ovrednotili z uporabo GH-IR modela. Sledila je termodinamska analiza adsorpcije z uporabo nelinearne Van ‘t Hoffove enačbe, najprej na bazičnih aminokislinah nato še na obeh modelnih proteinih. Na koncu smo pridobljene trende termodinamskih parametrov preverili še z izotermno titracijsko kalorimetrijo. Rezultati študije so jasno pokazali razlike v mehanizmu adsorpcije modelnih proteinov in s tem razlike v kromatografskih lastnostih uporabljenih ionskoizmenjevalnih kolon. Pri tem je najbolj izstopala kolona CIM SO4 2, ki je v primerjavi z ostalima kolonama izkazovala tudi multimodalne lastnosti. Cation-exchange chromatography is one of the principal techniques used for the separation and purification of proteins. In addition to electrostatic interactions, protein retention is often strongly influenced by the chemical nature of the ion-exchange ligand and by the properties of the column matrix. The aim of this study was to compare the performance of three different cation-exchange monolithic columns, CIM SO3, CIM SO4 1 and CIM SO4 2, which differed in the type of ion-exchange group, ligand density and matrix composition. The study focused on how these differences affect the adsorption mechanism of two model proteins, Cytochrome c and Lysozyme. The chromatographic behavior of the proteins was first investigated using linear salt-gradient elution. The ion-exchange process was subsequently characterized using the GH-IR model. This was followed by a thermodynamic analysis of adsorption based on the nonlinear Van ‘t Hoff equation, first applied to basic amino acids and then to both model proteins. Finally, the observed trends in thermodynamic parameters were further evaluated by isothermal titration calorimetry. The results clearly demonstrated differences in the adsorption mechanisms of the model proteins and, consequently, in the performance of the investigated ion-exchange columns. Among the studied supports, CIM SO4 2 showed the most distinct behavior and, compared with the other two columns, indicated a multimodal character. kationsko-izmenjevalna kromatografija monolitna kolona GH-IR model nelinearen Van ‘t Hoffov graf izotermna titracijska kalorimetrija ločba proteinov multimodalna kromatografija cation-exchange chromatography monolithic column GH-IR model nonlinear Van ‘t Hoff plot isothermal titration calorimetry protein separation multimodal chromatography true false false Slovenski jezik Angleški jezik Magistrsko delo/naloga 2026-07-02 14:10:03 2026-07-02 14:10:16 2026-07-31 11:07:52 0000-00-00 00:00:00 2026 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 25742 286567171 Kolenc_Eva_-_Studij_kromatografskega_obnasanja_modelnih_proteinov_na_razlicnih_kationsko-izmenje.pdf Kolenc_Eva_-_Studij_kromatografskega_obnasanja_modelnih_proteinov_na_razlicnih_kationsko-izmenje.pdf 1 627401B34233F1C2C7267E163EA5C2FB 854166a5fdb3b4fcb4c8247a364881ebb3853cc541d48c0241871cf31fecc408 e1665afc-760e-11f1-9b0d-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=236875 Fakulteta za kemijo in kemijsko tehnologijo 0 0 0