3.5. Analysis of TAG Composition with Silver Ion-High Performance Liquid Chromatography (Ag-HPLC)

TAGs of the lipids were separated using an Ag-HPLC (Younglin, Anyang, Korea) equipped with a silver ion column (Chromspher 5 lipids, 250 × 4.6 mm i.d., 5.0 μm, Varian, Middleburg, Netherlands) and an evaporative light scattering detector (ELSD, Sedex 75, Sedere, Alfortville, France). The temperature of the ELSD was set at 40 °C, and N₂ was used as a nebulizing gas at a pressure of 2.2 bar. Lipids were dissolved in chloroform: n-hexane (1:1, v/v) and 20 μL were injected into the HPLC system. The mobile phases were solvent A (n-hexane: isopropanol: acetonitrile = 100:0.1:0.1, v/v/v) and solvent B (n-hexane: iso-propanol: acetonitrile = 100:1:1, v/v/v) at a flow rate of 1.5 mL/min. TAG separation was started with solvent A for 5 min; the solvent ratio was increased to 80:20 (v/v) for 10 min, to 50:50 (v/v) for 10 min, held for 1 min, then returned to the initial condition of solvent A (100%), and finally held for 8 min. The TAGs were separated by the number of double bonds and the position of double bonds in the acyl chains, and the regioisomeric and enantiomeric isomers [37]. The individual TAG peaks in the HPLC chromatogram were identified by standard PPP, and designated by comparing the Ag-HPLC peaks of the OPO human milk fat substitute reported in Lee et al. [13], and TAG species reported in Hong et al. [38], which presented the Ag-HPLC chromatographs with the same methodology as the present study. Each TAG was presented with HPLC peak area or percentage (%) of total TAGs area.