Growth of two‐layer copolymer as the stationary phase with very high separation efficiency for separating peptides in capillary electrochromatography

Abstract

Open tubular CEC (OT‐CEC) column with a very high separation efficiency was prepared for peptides separation. A pretreated silica‐fused capillary was reacted with 3‐(methacryloxy) propyltrimethoxysilane followed by vinylbenzyl chloride and divinylbenzene to produce first thin monolithic monolayer. The second copolymer layer was formed on thin monolithic monolayer of the capillary by reversible addition‐fragmentation transfer polymerization of N‐phenylacrylamide and styrene. The key parameters including buffer pH value and organic modifier were systematically evaluated to provide the optimal chromatographic condition. The resultant OT‐CEC columns were validated by separating a synthetic mixture of peptides and cytochrome C tryptic digest in capillary electrochromatography. The number of theoretical plates as high as 2.4 million per column was achieved for synthetic mixture peptides. In addition, the fabricated OT‐CEC column also resolved more than 18 high‐efficiency digestion peptides from a mixture containing tryptic digest of cytochrome C. The column to column and inter‐ to intraday repeatabilities of OT‐CEC column through RSD% were found better than 3.0%, exhibiting satisfactory stability and repeatability of the two‐layer deposited OT‐CEC column. The results reveal that the open tubular capillary column modified with two‐layer copolymer shows the great prospect for the separation of proteins in capillary electrochromatography.

Recent applications and chiral separation development based on stationary phases in open tubular capillary electrochromatography (2019–2022)

“…The SP was poly (maleic anhydride-styrene- N -isopropylpropene) amide), which achieves baseline separation of three chiral drugs and three β-lactam antibiotics (cefradine, cephalexin, and amoxicillin) in human serum samples. The bilayer thin-film copolymer coating [ 45 ] prepared by an in-column successive reaction also shows excellent performance in the separation of peptides and trypsin digests.…”

“…Overall, we provide readers interested in OT-CEC with the most up-to-date overview of this field, and the related new SPs from 2019 to 2022 are listed in Table 1 [ 3 , [5] , [6] , [7] , [8] , [9] , [10] , [11] , [12] , [13] , [14] , [15] , [16] , [17] , [18] , [19] , [20] , [21] , [22] , [23] , [24] , [25] , [26] , [27] , [28] , [29] , [30] , [31] , [32] , [33] , [34] , [35] , [36] , [37] , [38] , [39] , [40] , [41] , [42] , [43] , [44] , [45] , [46] , [47] , [48] , [49] , [50] , [51] , [52] , [53] , [54] , [55] , [56] , [57] , [58] , [59] , [60] , [61] , [62] , [63] , [64] , [65] ]. PubMed and Web of Science were used to search the following characters (also their acronyms) alone or in combination: “Open tubular”, “Capillary electrochromatography”, “Chiral separation”, “Stationary phase”, and “Coating” for relevant literature to analyze recent OT-CEC developments.…”

Current trends in the development of polymer‐based monolithic stationary phases

“…Then, the second copolymer layer was formed on this thin monolithic mono- Reprinted with permission from Ref. [70] together with acryloyloxy-3-(methacryloyloxy)-2-propanol as a crosslinking monomer. After a thorough optimization of the composition of the polymerization mixture, the column was characterized using scanning electron microscopy.…”

“…68 Neequaye et al 69 A novel open tubular column applicable in CEC (OT-CEC) with a stable two-layer copolymer as the stationary phase was prepared for peptides separation and provided a very high separation efficiency. 70 At first, a very thin monolithic monolayer of vinylbenzyl chloride and divinylbenzene has been grafted onto the inner capillary wall. Then, the second copolymer layer was formed on this thin monolithic mono- Reprinted with permission from Ref.…”

Preparation of open tubular capillary column covalently coated with polystyrene sulfonate with 4,4′‐Azobis(4‐cyanopentanoyl chloride) as polymerization initiator for electrochromatographic separation of alkaloids, sulfonamides, and peptides

“…The prepared column was used in the separation of a small and medium-sized molecules. With the advancement in the field of peptidomics (study and quantification of endogenous peptides), the separation of peptides has become an essential aspect [8,[27][28][29][30][31][32][33][34][35], but it faces the challenge of adsorption on the capillary surface due to the interaction with silanol groups. To overcome this problem, the Azo-Co-PSS column is being used as a stationary phase and a repeatability study for three standard peptides for different parameters were performed.…”