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Separation of Tryptic Digested IgG with HPLC
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH).
2018 (English)Independent thesis Basic level (professional degree), 10 credits / 15 HE creditsStudent thesisAlternative title
Separation av trypsin-klyvt IgG med HPLC (Swedish)
Abstract [en]

The antibody immunoglobulin G (IgG) can be tryptically digested into smaller peptides. This study attempted to develop a separation method for those peptides using RP-HPLC with a C18 column at room temperature. Optimizing separation of trypsin cleaved cytochrome C was used as a guideline before analyzing IgG. The optimized analysis of Cytochrome C was performed at wavelength 280nm (UV) and methanol was used as an organic solvent in mobile phase (B). A fast gradient to 100% mobile phase B with low flow rate gave favorable results for cytochrome C. A slow gradient to 100% mobile phase B was suited for IgG separation. The optimized gradient elution of cytochrome C and IgG was performed at 0.3 and 0.8 ml/min, respectively.

Abstract [sv]

Antikroppen Immunoglobulin G (IgG) kan klyvas till peptider med enzymet trypsin. Under denna studie ska utvecklades en separationsmetod för dessa peptider med RP-HPLC. Separationen utfördes med en C18 kolonn i rumstemperatur. Först optimerades en separation av trypsin-klyvt cytokrom C vars optimerade parametrar sedan användes som grund för IgG-separation. Optimeringen utfördes vid våglängden 280 nm(UV) och metanol användes som organiskt lösningsmedel i mobil fas (B). En snabb gradient upp till 100% B med låg flödeshastighet gav mest gynnsamt resultat för cytokrom C. Separationen av IgG gynnades av ett högt flöde och en långsam gradient till 100% B. Den optimerade gradientelueringen för cytokrom C och IgG gjordes vid flödet 0.3 respektive 0.8 ml/min.

Place, publisher, year, edition, pages
2018.
Keywords [en]
IgG, trypsin, HPLC, gradient elution
National Category
Chemical Engineering
Identifiers
URN: urn:nbn:se:kth:diva-233869OAI: oai:DiVA.org:kth-233869DiVA, id: diva2:1243293
Educational program
Bachelor of Science in Engineering - Chemical Engineering
Available from: 2018-08-30 Created: 2018-08-30 Last updated: 2018-08-30Bibliographically approved

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