The Development of 2-Dimensional GC Method for Analysis of Natural Lipids Products from Enzymatic Microreaction

DOI: 10.5176/2251-2489_ BioTech14.22

Authors: Carla Villegas, Samuel M.Mugo, Sabiqah Tuan Anuar, Jonathan M. Curtis

Abstract: Difficulties were encountered in separating and quantifying low levels of neutral lipid classes present in crude reaction mixtures using one-dimensional GC and LC methods. These were shown to be due in part to matrix interferences along with widely differing response factors. The examples used are the products of lipid transformation reactions formed using a lipase-immobilized microreactor. It was shown that comprehensive two-dimensional gas chromatography with high temperature columns coupled to a flame ionization detector (GCxGC–FID) can successfully separate trace quantities of neutral lipid classes product of biotransformation from background components. The separation was performed using DB-5HT column (5{6e6090cdd558c53a8bc18225ef4499fead9160abd3419ad4f137e902b483c465} phenyl – methyl polysiloxane) in the first dimension and Rtx-65TG column (65{6e6090cdd558c53a8bc18225ef4499fead9160abd3419ad4f137e902b483c465} diphenyl - dimethyl polsiloxane) in the second dimension. The GCxGC method improves high temperature gas chromatography since the column bleed, which is a problem in 1D-GC, is totally resolved from the analytes. The GCxGC method was used to separate and quantify a mixture of ethyl oleate, monoolein, diolein and triolein over the concentration range of 0.025 to 0.125 mg/mL. Preliminary method validation indicated good linearity, precision and repeatability along with stable retention times in both dimensions over the course of a month. The limit of quantification expressed as on-column amounts were in the range of 7 pg for ethyl oleate, 500 pg for mono- and di- olein and 70 pg for triolein. Thus, in addition to other advantages, the GC x GC method is 2 or more orders of magnitude more sensitive than GC-FID or LC with evaporative light scattering detection and the effective separation allows quantification of trace lipid classes in the presence of more abundant components.

Keywords: Comprehensive two-dimensional gas chromatography, neutral lipids, enzymatic microreactor, biotransformation, lipid class separation