Clarus 680

Capillary high-resolution gas chromatography (HR-GC) with a CP-Sil 88 CB column was used to analyze the fatty acids in all sample extracts. The GC column was installed on a PerkinElmer gas chromatograph CLARUS 680 equipped with a flame ionization detector and split injection, as previously described [53 (link)]. In brief, hydrogen was employed as the carrier gas at a flow rate of 1 mL × min⁻¹ while the split ratio was 1:20, with the injector and detector adjusted at 260 °C and 280 °C, respectively. The GC oven temperature programme was 150 °C for 5 min; 2 °C/min heating until 200 °C and held for 10 min; and 1 °C/min heating until 225 °C and held for 20 min. To calibrate the reference standard mixture “Sigma FAME”, the methyl esters of C18:1cis-11, C22:5n-3, and C18:2cis-9, and trans-11, C22:4n-6, and C18:4n-3 were employed. After GC analysis of 5 samples, the 5-point calibration of single fatty acids assessed were ranged between 16 and 415 µg/mL. Fatty acid concentrations were displayed in milligrams per gram of egg yolk.

FT-IR/NIR Spectroscopy Frontier (Perkin Elmer, Waltham, MA, USA) and Bruker AC 500 MHz (Bruker Co., Billerica, MA, USA) were used to analyze the chemical structure of synthesized products. To obtain the FT-IR spectra, samples (1–2 mg) were prepared by mixing them with dried KBr (100–200 mg) and then pressing the mixture into a pellet. The measurement acquired in range of 4000–500 cm⁻¹ with a resolution of 4 cm⁻¹. Gas chromatography (GC) (PerkinElmer Clarus 680) analysis of final product was carried out. Synthesized mPEG-G3.5 was dissolved in diethyl ether as solvent for GC run. TEM (JEM-1400, Tokyo, Japan) with an accelerating voltage of 100 kV was used to depict the size and morphology of the products. Sample at a concentration of 1 mg/mL was placed on a carbon-copper grid (300-mesh, Ted Pella Inc., Redding, CA, USA) and air-dried for 10 min before the measurement. Zeta potential and hydrodynamic diameter of the products were determined by Nano Particle Size SZ-100 (Horiba, Kyoto, Japan). Samples were prepared with deionized water at a concentration 1 mg/mL, filtered (pore size 0.45 μm), and sonicated for 5 min. Each sample was measured 3 times.

GC-MS^{24 (link),25 (link)} is one of the commonly used instruments for identifying and separating organic molecules. It is a typical chemical analysis method for accurate quantitative and qualitative analysis. In this case, GC-MS was used to observe and compare the EEC after pure EEC and UV light. The experiments were performed using a PerkinElmer Clarus 680 gas chromatograph (GC) connected to a PerkinElmer Clarus 600T mass spectrometry (MS). The relative affinity of the stationary phases of the different molecules in the sample would promote the separation of the molecules. The column used in the GC experiment was Elite-624, which had a length and an inner diameter of 30.0 m × 0.25 mm and a film thickness of 1.4 μm. The molecular effluent of the column was captured, and the electron ionization (70 eV) fragment was detected using their mass to charge ratio. A helium flow rate of 1.0 mL/min was used as the carrier gas for the column.

The liver tissue was lysed in a methanol solution (1:10) with ultrasound and mixed with chloroform solution. After adding distilled water, the mixture was separated into two layers after centrifuging at 3000 rpm, at 4 °C, for 10 min. The upper layer containing the organic solvent was separated, and ethanol plus triton X mixture as emulsifiers (1:1 v/v) was added to the organic solvent part. The triglyceride contents in the emulsifier layer were measured using a triglyceride kit (Asan Pharmaceutical, Korea).
The liver or skin tissues were lysed in lysis buffer (1:10) with ultrasound. A mixture of cold 0.25N-HCl, 15% trichloroacetic acid, and 0.38% 2-thiobarbituric acid was added to the lysed liver tissue. The mixture was placed in a 100 °C incubator for two hours. After cooling, the supernatants were separated with centrifugation at 5000 rpm at 4 °C for 10 min, and their optical density was detected at 532 nm in a spectrophotometer (Perkin-Elmer, Boston, MA, USA). The contents of 2-thiobarbituric acid reactive substances (TBARs) were measured in the liver and skin tissue extract using TBARS assay kit (Abcam, Cambridge, UK).
The short-chain fatty acid (SCFA) contents of the serum from the portal vein were analyzed by gas chromatography (Clarus 680, PerkinElmer, Boston, MA, USA); 1 mM acetate, propionate, and butyrate (Sigma, St. Louise, MO, USA) were used as standards [62 (link)].

A polar column (DB-WAX, 30 m 0.25 mm 0.25 m, Agilent Technology, Santa Clara, CA, USA) was used in a PerkinElmer GC–MS (GC, Clarus 680; MS, Model SQ8C) using helium as the carrier gas at a follow rate of 1 mL/min. The initial oven temperature was held at 35 °C for 5 min, then ramped up to 140 °C at 4 °C/min and kept for 5 min, before being elevated to 230 °C at 10 °C/min and kept for 5 min. An MS detector was used with a mass scan range of 33–450 amu (m/z) and a 70 eV electron ionization (EI) voltage. Both the ion source and line transfer were adjusted at 250 °C.

The contents of AA, PA, IBA, BA, IVA, and VA were determined by GC-FID (Clarus® 680, PerkinElmer, United States). Detailed experimental steps are provided in Supplementary Method S2.