Optimizacija SEC metode za karakterizacijo svile

Piciga, Sara (author), Kralj Cigić, Irena (mentor), Kraševec, Ida (comentor)

V diplomskem delu sem optimizirala metodo velikostno izključitvene kromatografije (SEC) za določanje proteina fibroina, ki je glavna komponenta svile. Analiza s SEC metodo omogoča vpogled v porazdelitev molskih mas fibroina, kar je pomembno za karakterizacijo svile, ki je pomembna tako za številne sodobne aplikacije svile, na primer v biomedicini, kot tudi za razumevanje razgradnje svile v predmetih kulturne dediščine. Prvi korak diplomskega dela je bila izbira ustreznega topila za redčenje standarda fibroina, ki je bilo hkrati primerno tudi kot mobilna faza za SEC . Preizkusila sem različne pufre in dodatke soli. Kot najustreznejše topilo se je izkazal 0,1 M citratni pufer s pH 4,5 in dodatkom 0,5 M KCl. Sledila je optimizacija SEC metode, ki sem jo najprej izvedla na BSA, nato še na fibroinu. Optimizirala sem detekcijo, kolone, analitske pogoje in mobilno fazo in kot najustreznejša se je izkazala postavitev z eno predkolono in eno kolono ter metodo dolžine 40 minut, s pretokom 0,5 mL/min, volumnom injiciranja 10 μL in valovno dolžino detekcije 280 nm. Za določitev molske mase fibroina sem izvedla umeritev s proteini z znanimi molskimi masami. Ker nisem imela na voljo proteina z dovolj visoko molsko maso, je bila umeritev primerna le za približno oceno molske mase standarda fibroina, ki je znašala 123,5 kDa. Na koncu sem optimizirano metodo uspešno preverila še na dveh realnih vzorcih svile.

• Language: Slovenian
• Keywords: svila, fibroin, SEC, optimizacija
• Work type: Bachelor thesis/paper
• Typology: 2.11 - Undergraduate Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2025
• PID: 20.500.12556/RUL-169883
• COBISS.SI-ID: 239747075
• Publication date in RUL: 13.06.2025

Secondary language

• Language: English
• Title: Optimization of SEC method for silk characterization

Abstract

In this thesis, I optimized the size exclusion chromatography (SEC) for determination of protein fibroin, the main component of silk. SEC analysis enables insight into the molecular weight distribution of fibroin, which is important for characterization of silk, which is important both for numerous modern applications of silk, such as in biomedicine, and for understanding the degradation of silk in cultural heritage objects. The first step of the thesis was choosing a suitable solvent for diluting the fibroin standard, which also had to be suitable to use as the mobile phase for SEC. I tested various buffers and salt additives. The most suitable solvent proved to be 0.1 M citrate buffer with pH 4.5 containing 0.5 M KCl. This was followed by the optimization of the SEC method, initially using BSA, and later fibroin. I optimized detection, columns, analytical conditions and mobile phase. The most suitable configuration was a pre-column and one column with a method length of 40 minutes, a flow rate of 0.5 mL/min, an injection volume of 10 μL and a detection wavelength of 280 nm. To determine the molar mass of fibroin, I performed a calibration with proteins with known molar masses. As no protein with a sufficiently high molar mass was available, the calibration was only useful for an approximate estimation of molar mass of fibroin, which was 123.5 kDa. Finally, I successfully validated the optimized method on two real silk samples.

• Keywords: silk, fibroin, SEC, optimization