Separation of peptides on superficially porous particle based macrocyclic glycopeptide liquid chromatography stationary phases: consideration of fast separations

A stationary phase was prepared by chemical derivatization of the support particles with a layer of copolymer composed of styrene and N‐phenyl acrylamide. Silica monolith particles of ca. 2.6 µm (volume‐based average) have been prepared as the support particles by sol‐gel reaction followed by differential sedimentation. The particles were reacted with 3‐chloropropyl trimethoxysilane followed by sodium diethyldithiocarbamate to introduce an initiator moiety. Then, the copolymer layer was immobilized via reversible addition‐fragmentation transfer polymerization. The resultant phase was packed in glass‐lined stainless‐steel micro‐columns (1 x 150 mm) and evaluated for the separation of a mixture composed of five peptides (Trp‐Gly, Thr‐Tyr‐Ser, angiotensin I, isotocin and bradykinin). The effect of monomer mixing ratio (styrene versus N‐phenyl acrylamide) on the chromatographic separation efficiency of the stationary phase was examined. A number of theoretical plates (N) as high as 33 600 plates/column (224 000 plates/m, 4.46 µm plate height) was achieved using the column packed with the optimized stationary phase. The column‐to‐column reproducibility based on three columns packed with three different batches of stationary phase was found satisfactory in separation efficiency, retention factor, and asymmetry factor.

Section: Introductionmentioning

confidence: 99%

Styrene‐N‐phenylacrylamide co‐polymer modified silica monolith particles with an optimized mixing ratio of monomers as a new stationary phase for the separation of peptides in high performance liquid chromatography

Kim

et al. 2019

“…The advantages and application potential of mixed‐mode reversed phase/positively charged repulsion stationary phase were explored for intact protein separation . Teicoplanin and vancomycin chiral selectors were attached covalently to the surface of 2.7 µm superficially porous particles, resulting in good performance in separation of pharmaceutically important peptides .…”

Section: Introductionmentioning

confidence: 99%

“…The best result may be found in ref. where the separation efficiency of [Arg8] vasopressin was 110 000 plates/m with the retention time of about 5 min at a flow rate of 1 mL/min in 17:83, v/v acetonitrile/50 mM ammonium formate for the 4.6 × 100 mm column packed with a 2.7 µm core/shell teicoplanin stationary phase (11 000 plates/column).…”

Section: Introductionmentioning

confidence: 99%

An optimized mixed‐mode stationary phase based on silica monolith particles for the separation of peptides and proteins in high‐performance liquid chromatography

Ali

Kim

et al. 2019

A phase with both hydrophobic and hydrophilic functionalities has been synthesized by modification of ground silica monolith particles with C18 and 1‐[3‐(trimethoxysilyl)propyl] urea ligands. A series of phases was prepared by changing the ratio of the two ligands to determine the optimal ratio in view of separation efficiency. The resultant optimized stationary phase was packed in narrow‐bore glass‐lined stainless‐steel columns (1 × 300 mm and 2.1 × 100 mm) and used for the separation of synthetic peptides and proteins. The average numbers of theoretical plates (N) of 52 100/column (174 000/m, 5.75 µm plate height) and 35 500/column (118 000/m, 8.47 µm plate height) were achieved with the 300 mm column at a flow rate of 25 µL/min (0.86 mm/s) in 60:40 v/v acetonitrile/30 mM aqueous ammonium formate for the mixture of peptides (Thr‐Tyr‐Ser, Val‐Ala‐Pro‐Gly, angiotensin I, isotocin, and bradykinin) and for the mixture of proteins (myoglobin, human serum albumin, and insulin), respectively. Fast analysis of the peptides and proteins was also carried out at a flow rate of 0.9 mL/min (6.88 mm/s) with the 100 mm column and all the analytes were eluted within 2 min with good separation efficiency.

“…In comparison to the RPLC and other conventional chromatographic modes, the mixed‐mode chromatography (MMC) stationary phases are considered to be effective in the achievement of better separation selectivity for polar species including peptide and proteins. MMC reports to be one of a promising approach for the separation and the detailed analysis of different classes of compounds [3], including the separation of peptides and proteins [4,5].…”

Section: Introductionmentioning

confidence: 99%

“…An average separation efficiency of 224 000 plates/m has been reported for a mixture of Trp‐Gly, Thr‐Tyr‐Ser, angiotensin I, isotocin, and bradykinin in a very recently published article [2], where the stationary phase is somewhat similar to MMC stationary phases. Another reference [5] reports separation efficiency of 110 000 plates/m for the vasopressin. Some of the research reports on the separation efficiency calculated from the documented chromatograms under isocratic elution mode are much inferior to that of the previous study [2].…”

Section: Introductionmentioning

confidence: 99%

Particle packed mixed‐mode chromatographic stationary phase for the separation of peptide in liquid chromatography

Ali

Cheong

Rafique³

et al. 2021

A particle‐based stationary phase has been prepared for the separation of five synthetic peptides and a mixture containing tryptic digest of cytochrome C in liquid chromatography. Particles originating from silica monolith were differentially sedimented to obtain 1–2 μm particles. A stationary phase was achieved by the coating of poly(styrene‐methacrylic acid‐N‐phenylacrylamide) copolymer onto the particles via reversible addition‐fragmentation chain transfer polymerization reaction. Stainless steel column (30 cm long and 1 mm internal diameter) was packed with stationary phase. Very high separation efficiency (ca. 351 000 plates/m) was achieved for five commercial peptides with a percent relative standard deviation of less than 1%. Protocol for the synthesis and modification of silica monolith particles has been well optimized with a good reproducibility both in particle and pore size. The column resolved about 21 peptide components from a mixture containing tryptic digest of cytochrome C, under the elution conditions of acetonitrile/15 mM ammonium format (65/35 v/v%) with a flow rate of 28 μL/min.