Sorbent for HPLC based on copolymer of styrene and divinylbenzene with gold nanoparticles stabilized by eremomycin
Authors: Prosuntsova Daria S. , Ananieva Irina A. , Moskaleva Tatiana A. , Shpigun Oleg A. 
Published: August 2024

In liquid chromatography, the use of new hybrid materials with nanoparticles as stationary phases offers an alternative to the commonly used silica-based phases. This paper presents a study of polymer nanohybrid sorbent with macrocyclic antibiotic. Eremomycin-stabilized gold-coated polystyrene-divinylbenzene (PS-DVB) particles are synthesized. PS-DVB-Au-eremomycin sorbent is characterized by atomic emission spectroscopy and elemental analysis. The concentration of gold nanoparticles (GNPs) immobilized onto PS-DVB matrix is approximately equal to 3.5∙1014 particles per gram. The immobilization of eremomycin provides bonding density of 20 µmol/g. Sorbent has a mesoporous structure according to the low-temperature nitrogen adsorption results. The functional surface area of the obtained sorbent compared to unmodified PS-DVB increases by 30%, despite the immobilization of large molecules of antibiotic, which is an important benefit of GNPs adsorption.
The obtained sorbent is studied as a stationary phase for high performance liquid chromatography (HPLC). A detailed study of the retention mechanisms of profens and β-blockers on PS-DVB-Au-eremomycin is presented. The retention factors are obtained at varied pH, organic solvent content, and ionic strength of the mobile phase. The retention factors of profens at pH 4.0 are low, however, an increase in retention with an increase in acetonitrile content from 20 % to 80% is observed. This behavior could be explained by a growing contribution of hydrophilic interactions into retention. The best separation of the mixture of profens is obtained at 80 vol. % acetonitrile. At pH 9.9, the presence of hydrophobic interactions was indicated by the decrease of retention with an increase of organic content. The best separation of the four profens is achieved at 20 vol. % of acetonitrile. Retention order: indoprofen < ketoprofen < ibuprofen < flurbiprofen, corresponds to the increase in the hydrophobicity of the substances. This indicates to a typical reversed-phase mechanism. At pH 6.2, the retention order of analytes changes, which means a change in the retention mechanism; an increase in the retention times of analytes is also observed. Retention is determined by a mix of ion exchange and classical adsorption mechanisms. The retention of profens decreases with an increase in the concentration of the buffer solution, which also indicates the presence of ionic interactions.
Retention of β-blockers increases with an increase in acetonitrile content. This effect seems to match well with an electrostatic repulsion hydrophilic interaction retention mechanism. However, a dependence between hydrophobicity and retention of β-blockers was also found. At pH 9.9, at which β-blockers are adsorbed as neutral molecules, this dependence is most pronounced. This indicates the presence of hydrophobic interactions. When pH is lowered to 6.2, the retention order of β-blockers changes, which indicates the competition of different mechanisms of interaction between the sorbate and the sorbent. For β-blockers with an increase in the concentration of the buffer solution the retention increases and the selectivity also increases. The obtained results confirm early observations that electrostatic interactions play a significant role in the retention mechanism. The higher ionic strength of the eluent results in the suppression of such interactions and may influence separation selectivity.
The enantioselective properties of the sorbent have been demonstrated. The best separation of profene enantiomers is observed when the content of acetonitrile in eluent is 90-95% and at a neutral pH. Enantiomers of β-blockers separate under conditions of neutral pH and the predominance of the water component in eluent. To expand the scope of the chiral sorbent, the separation of enantiomers of benzoxycarbonyl- (CBZ), benzoyl-, tert-butoxy- (BOX) derivatives of amino acids on an experimental sorbent were also studied. Separation of the enantiomers of some CBZ and benzoyl derivatives has been obtained.
The column with PS-DVB-Au-eremomycin was used to analyze a drug "Ketonal" by HPLC, the content of two enantiomers of ketoprofen in its composition was confirmed.
A mixed-mode retention mechanism with a combination of electrostatic, hydrophobic, and hydrophilic interactions is realized on the synthesized nanohybrid stationary phase. The hydrophobic interactions dominated the retention of the profens at alkaline pH. Electrostatic and hydrophilic retention appears to play a role, under acidic and neutral conditions, in the retention of profens on the stationary phase. The gradual increase in the retention of β-blockers with the addition of acetonitrile into the mobile phase indicates HILIC behavior. Hydrophobic interactions are not entirely dominant on the investigated stationary phase, but they also play an essential role in retention. It has been shown that the retention factors of profens and β-blockers correlated with the polarity. The obtained sorbent showed enantioselectivity to various groups of organic substances, which demonstrates its potential as a chiral stationary phase.