Preparation of mixed-mode stationary phase for separation of peptides and proteins in high performance liquid chromatography

Sarah Alharthi,Fazal Mabood,Aliya Ibrar,Sobia Nisa,Ashraf Ali,Bashir Ahmad,Muzaffar Iqbal

doi:10.1038/s41598-022-08074-7

Sarah Alharthi, Fazal Mabood + Show 5 more

Open Access

https://doi.org/10.1038/s41598-022-08074-7

Copy DOI

Abstract

Porous silica particles were prepared by sol–gel method with some modification to get wide-pore particles. These particles were derivatized with N-phenylmaleimide-methylvinylisocyanate (PMI) and styrene by reversible addition fragmentation chain transfer (RAFT) polymerization to prepare N-phenylmaleimide embedded polystyrene (PMP) stationary phases. Narrow bore stainless steel column (100 × 1.8 mm i.d) was packed by slurry packing method. The chromatographic performance of PMP column was evaluated for the separation of synthetic peptides mixture composed of five peptides (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, Leucine enkephalin) and tryptic digest of human serum albumin (HAS) respectively. Number of theoretical plates as high as 280,000 plates/m were obtained for peptides mixture at optimum elution condition. Separation performance of the developed column was compared with commercial Ascentis Express RP-Amide column and it was observed that separation performance of PMP column was better than commercial column in terms of separation efficiency and resolution.

Highlights

Porous silica particles were prepared by sol–gel method with some modification to get wide-pore particles
polyethylene glycol (PEG), urea, acetic acid, trimethoxy orthosilicate (TMOS), Chlorotrimethylsilane(TMCS), trypsin, human serum albumin (HSA), ammonium chloride, urea, hexamethyldisilazane (HMDS), methacryloyl chloride (MC), styrene, 4-hydroxy-TEMPO, benzoyl peroxide (BPO), HPLC grade acetonitrile (ACN), methanol, 2-propanol, and acetone were purchased from Sigma–Aldrich
The chromatographic performance of phenylmaleimide embedded polystyrene (PMP) column for peptides mixture is excellent in terms of separation efficiency and resolution

Summary

Introduction

Porous silica particles were prepared by sol–gel method with some modification to get wide-pore particles These particles were derivatized with N-phenylmaleimide-methylvinylisocyanate (PMI) and styrene by reversible addition fragmentation chain transfer (RAFT) polymerization to prepare N-phenylmaleimide embedded polystyrene (PMP) stationary phases. Abbreviations PMP N-phenylmaleimide embedded polystyrene RP Reversed phase HILIC Hydrophilic interaction liquid chromatography ODS Octadecyl-modified silica RAFT Reversible addition fragmentation chain transfer polymerization SI-ATRP Surface initiated-atom transfer radical polymerization DBTD Dibutyltin dichloride HAS Human serum albuim PMCP Phenylmaleimide-methylvinylisocyanate copolymer. Reverse phase liquid chromatography (RP-LC) is widely used for the purification and separation of peptides mixture using octadecyl-modified silica (ODS) as a stationary p hase[11–13]. Polar embedded columns such as amide embedded C18 columns are commercially available under the trade name Ascentis Express RP-Amide column, but these columns have only been used for the analysis of a mines[33]

Methods

Results

Conclusion