Separation of peptides with an aqueous mobile phase by temperature‐responsive chromatographic column

Abstract

Peptide separation technology is significant and is still an analytical challenge in proteomic studies. We report a simple preparation method for poly(N-isopropylacrylamide) grafted silica through the copolymerization of N-isopropylacrylamide with acetyl moieties immobilized on the silica surfaces. Differential scanning calorimetry results indicated that the prepared silica exhibited a sharp phase transition at 35.03°C. Silica grafted with poly(N-isopropylacrylamide) was evaluated as a temperature-responsive chromatography medium for the separation of peptides using 0.2 M NaCl solution as a mobile phase. Results indicated that at 10°C, the peptides were not resolved, but baseline separation with prolonged retention time at 50°C was attained. Particularly, a mixture of four peptides was efficiently separated within 8 min. The theoretical plate number of every peptide was more than 2500, and the resolutions were more than 3.40. The increased selectivity of the temperature-responsive column resulted from the temperature-modulated hydrophobic interaction with peptides. The retention times of these peptides were related to their hydrophobicities. This protocol provided a reliable set of chromatographic tool usable across all research and development applications that required isolation and analysis of peptides. It may represent a step forward in the complex analysis of hydrophobic and other proteins.

Effect of the grafting ratio of poly(N‐isopropylacrylamide) on thermally responsive polymer brush surfaces

“…We prepared four kinds of 2-bromoisobutyrylbromide functionalized silica beads according to a previous report [7,8,11,15]. 3-Aminopropyl silica (2.2 g) was placed into a solution of triethylamine (1.5 mL) and anhydrous tetrahydrofuran (20 mL).…”

“…The separation effect of grafted stationary phase was significantly enhanced and retention time was extended with the increase in temperature . The separation of compounds on the thermally responsive column could be performed with an aqueous mobile phase without any organic solvent , which will be beneficial for reducing the environmental pollution.…”

“…The separation of compounds on the thermally responsive column could be performed with an aqueous mobile phase without any organic Abbreviations: LCST, lower critical solution temperature; NIPAAm, N-isopropylacrylamide; PMDETA, N,N,N',N'',N'',-pentamethylenetriamine; TGA, thermogravimetric analysis Conflict of Interest: The authors have declared no conflict of interest. solvent [13][14][15][16], which will be beneficial for reducing the environmental pollution.…”

Enrichment of adenosine using thermally responsive chromatographic materials under friendly pH conditions

“…Recently, thermally responsive materials have found wide applications in the separation of biomolecules. Poly( N‐ isopropylacrylamide) (P(NIPAAm)) is a well‐known thermoresponsive polymer, which has a lower critical solution temperature (LCST) of 32°C and it exhibits a reversible change in solubility in response to changes in temperature . Copolymerization of NIPAAm with boron‐containing monomers such as 4‐vinylphenylboronic acid (VPBA) and AAPBA exhibit pH, glucose, and thermal sensitivities.…”

Microfluidic chip with thermoresponsive boronate affinity for the capture–release of cis‐diol biomolecules

“…Poly( N ‐isopropylacrylamide) (PNIPAAm) is the most popular thermally responsive polymer , and phenylboronic acid (PBA) can reversibly and covalently bind with cis ‐diol‐containing biomolecules , such as nucleotides , carbohydrates , and glycoproteins . The P(NIPAAM‐PBA) copolymer not only possesses the properties of pH and glucose sensitivity, but also possesses the property of temperature sensitivity .…”