Development of graphene oxide and derivative HPLC stationary phases for separation of β-carotene and lutein

In this thesis study; it was aimed to synthesis and characterization of graphene oxide, Si-GO and Si-GO-C18 materials, to development of methods for the separation of β-carotene and lutein molecules, and validate these methods. Graphene oxide (GO), synthesized using the Hummers method modified with H2SO4 and H3PO4, was first chemically bonded to aminopropyl silica to obtain the Si-GO stationary phase, and then the Si-GO-C18 stationary phase was obtained by bonding C18 to this stationary phase. Characterization of the synthesized GO, Si-GO and Si-GO-C18 materials was carried out by UV-vis, FTIR, TEM and SEM analyses. Highly oxidized GO nanoparticles were subjected to FTIR analysis and -OH and -COOH groups, which give hydrophilicity to graphene oxide, and C=C, C=O and epoxide (C–O–C) peaks were detected. It was observed that some of the -OH and -COOH peaks in GO decreased and the peptide bond was formed. Additionally, -CH vibrations indicating C18 binding on GO were detected in the Si-GO-C18 spectrum. It was observed that the TEM and SEM images of the obtained GO and stationary phases also supported the FTIR analysis. Both of the obtained Si-GO and Si-GO-C18 stationary phases were filled into different columns at high pressure. The columns were prepared for analysis by washing with methanol, pure water and acetonitrile, respectively. Both isocritical and gradient separations were performed on the Si-GO column to separate β-carotene and lutenin, and it was observed that the desired separations could not be achieved. A chemometric design was made to improve the separations, and the resolution and teoretical plates were calculated by creating 21 different methods. The optimum method parameters were determined to be IPA mobile phase composition 25%, flow rate 1.8 mL/min and temperature 30°C by using of the desirability function (D) of the chemometric design. The % relative standard deviation, % recovery, LOD, LOQ and R2 values were calculated for validation studies of the method whose optimum parameters were determined. It was found that the %RSD, the recovery, LOD and LOQ values were 3.40 and 2.76, 90% and 93%, 0.27 and 0.34 mg/L, and 0.09 and 0.11 mg/L, respectively. Both isocritical and gradient separations were performed in method determination studies using the Si-GO-C18 column. The desired result could not be obtained from isocritical studies, and it was determined that Method 6 was suitable for the separation of β-carotene and lutene from gradient separations by calculating the resolution and teoretical plates. It was found that the %RSD, the recovery, LOD and LOQ values were 0,86 ve 1,09, 95% and 80%, 0.26 and 0.27 mg/L, and 0.80 and 0.82 mg/L, respectively.