Chromatographic and Electrophoretic Studies: Separation of Biofluid Components, Nanomaterials, and Evaluation of Separation Media
Tirado González, Karina M.
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Untargeted analysis of complex biological mixtures is a demanding and challenging task. High performance liquid chromatography (HPLC) and mass spectrometry (MS) were used to investigate the composition of human ascites, a biological fluid with a composition that is not fully known. Several chromatographic fractions of ascites were isolated by means of reversed phase liquid chromatography (RPLC). Of these, the most polar fraction was further studied by hydrophilic interaction liquid chromatography (HILIC). Seven of the components that comprised this fraction were identified (i.e, proline, betaine, pyroglutamic acid, stachydrine, (3-carboxypropyl) trimethylammonium, carnitine, and glucose). The studies set the foundation to further study the composition of human ascites. As the sample complexity increases, new materials are desired for more effective ways to perform sample preparation procedures. Bare silica and oxygenated nanodiamonds were assessed as sorbents for the extraction of nucleosides and nucleotides in the HILIC mode. After optimization, bare silica was showed to be a viable option for the sample cleanup of nucleotides with recoveries ≥ 69% in a lysate matrix; recoveries for nucleosides were poor. The evaluation of oxygenated nanodiamonds sorbent material indicated their potential for nucleoside extraction, but deficient for nucleotide extraction. The two adsorbent materials showed to be complementary. As a new chromatographic material becomes available, it is important to evaluate its performance. Several HPLC columns (i.e., Acquity BEH C 18 , Kinetex EVO C 18 , Cortecs C 18 , and Kinetex C 18 ) were evaluated under different conditions to assess their silanol activity using two basic compounds as probes (i.e. amitriptyline and dextromethorphan). It is shown that using dextromethorphan as a probe compound results in a stronger interaction with surface silanols than amitriptyline. Peak asymmetry, which is indicative of silanol activity, becomes more prominent when using acetonitrile instead of methanol in the mobile phase. RPLC columns containing silica hybrids showed less silanol activity than the traditional silica supports. The trend in asymmetry factor for the separation of basic compounds showed to be equal for the columns tested here (i.e., Acquity BEH > Kinetex EVO > Cortecs > Kinetex) regardless of the mobile phase used. Carbon dots (CDs) are an emerging class of nanomaterial with dimensions of less than 10 nm. CDs are produced in bulk and usually separated by low-resolution methods, still rendering complex mixtures. We explored the separation of carbon dots mixtures by means of size exclusion HPLC using two chromatographic columns with different pore sizes (i.e., 300 Å and 150 Å). While the column with larger pore size provided the expected size exclusion mechanism, the column with lower porosity further enhanced the separation of various components in the CDs mixture. CDs were also separated by capillary electrophoresis (CE), which allowed the monitoring of different photoluminescent species produced as a function of precursors’ concentration. It was observed that negatively charged species were the major product when citric acid, one of the precursors, exceeded the amount of amine (the second precursor) present in the reaction. At equal or greater amounts of the amine precursor in the reaction mixture, the main product was neutral under the conditions studied.