Application of silica-based hyper-crosslinked sulfonate-modified reversed stationary phases for separating highly hydrophilic basic compounds

Abstract

The separation and determination of hydrophilic basic compounds are of great importance in many fields including clinical and biological research, pharmaceutical development and forensic analysis. However, the most widely used analytical separation technique in these disciplines, reversed-phase liquid chromatography (RPLC), usually does not provide sufficient retention for several of the important classes of highly hydrophilic basic compounds including catecholamines, many drug metabolites and many drugs of abuse. Commonly eluents having little or no organic modifier and/ or strong ion pairing agents must be used to achieve sufficient retention and separation. Use of highly aqueous eluents can lead to column failure by dewetting, resulting in poor retention, selectivity, reproducibility and slow recovery of performance. The use of a strong ion pairing agent to increase retention renders the separation incompatible with mass spectrometric detection and complicates preparative separations. This paper describes the successful applications of a novel type of silica-based, hyper-crosslinked, sulfonate-modified reversed stationary phase, denoted as − SO 3 -HC-C 8 -L, for the separation of highly hydrophilic cations and related compounds by a hydrophobically assisted cation-exchange mechanism. Compared to conventional reversed-phases, the − SO 3 -HC-C 8 -L phase showed significantly improved retention and separation selectivity. Concurrently, due to the presence of both cation-exchange and reversed-phase retention mechanisms and the high acid stability of hypercrosslinked phases, the separation can be optimized by changing the type or concentration of ionic additive or organic modifier, and by varying the column temperature. In addition, gradients generated by programming the concentration of either the ionic additive or the organic modifier can be applied to reduce the analysis time without compromising resolution. Furthermore, remarkably different chromatographic selectivities, especially toward cationic solutes, were observed upon comparison of the − SO 3 -HC-C 8 -L phase with conventional reversed-phases. We believe that the combination of these two types of stationary phases will be very useful in two-dimensional liquid chromatography.

Strong cation exchange column allow for symmetrical peak shape and increased sample loading in the separation of basic compounds

“…Silica-based SCX columns, either commercially available or self made, are now emerging in literatures [20][21][22]. However, the SCX mode is still adapted mostly in separations of biomacromolecules like peptides and proteins [23][24][25][26][27], and only a few used for analysis of basic drugs or alkaloids [16,[18][19][20]22,28], possibly due to low efficiency and asymmetric peak shape of bases. Furthermore, few silica-based SCX columns which allow for symmetrical peak shape and high sample loading in the chromatographic preparation of basic compounds was reported.…”

Fases estacionárias modernas para cromatografia líquida de alta eficiência em fase reversa

“…68 Conforme mencionado, a seletividade da FE é dependente de um conjunto de fatores, entretanto, o principal motivador no desenvolvimento de FE com diferentes seletividades está relacionado com a dificuldade encontrada nas separações de compostos polares, que engloba a maioria dos compostos básicos, por CLAE-FR empregando FE puramente alifáticas. [69][70][71] Esta dificuldade é decorrente de dois fatores principais: estes compostos são retidos insuficientemente na FE (que possui características hidrofóbicas); estes compostos interagem fortemente com os grupos silanois residuais da FE (que possuem características ácidas) resultando em picos cromatográficos com cauda acentuada, levando, inclusive, a retenções irreversíveis na fase estacionária. A retenção dos compostos polares pode ser aumentada com o uso de FM com altas quantidades de água, porém, o uso de FM com porcentagens de água acima de 95% leva à falência da coluna cromatográfica, além de ser incompatível com a detecção por EM.…”

Selective separation and purification of highly polar basic compounds using a silica-based strong cation exchange stationary phase

“…Salts are the most commonly used mobile phase additives in the cation exchange mode [15]. They can act as a buffer to affect the dissociation of basic compounds and control pH of mobile phase, and create the ion-association complexes.…”

“…Evans did a very good job to apply ion-exchange electrochromatography for basic compound separation [22]. Luo et al [15] developed an SCX based method to separate highly polar basic compounds. Several highly polar compounds, such as dopamine and epinephrine, were well separated by this method.…”

“…Strong cation-exchange (SCX) mode is an alternative to reversed phase and HILIC mode for high polar basic compound separation [15][16][17][18]. Although SCX mode is widely used for the analysis of biomacromolecule [19,20] and inorganic ions [21], only a few applications for the analysis of small molecule were reported.…”