Performance of short-chain alcohols versus acetonitrile in the surfactant-mediated reversed-phase liquid chromatographic separation of β-blockers

Organic solvents are traditionally added to micellar mobile phases to achieve adequate retention times and peak profiles, in a chromatographic mode which has been called micellar liquid chromatography (MLC). The organic solvent content is limited to preserve the formation of micelles. However, at increasing organic solvent contents, the transition to a situation where micelles do not exist is gradual. Also, there is no reason to neglect the potentiality of mobile phases containing only surfactant monomers instead of micelles (high submicellar chromatography, HSC). This is demonstrated here for the analysis of β-blockers. The performance of four organic solvents (methanol, ethanol, 1-propanol, and acetonitrile) was compared in mobile phases containing the anionic surfactant sodium dodecyl sulphate in the MLC and HSC modes. The association of the organic solvent molecules with micelles gives rise to a significant loss in the elution strength of the organic solvent; whereas upon disruption of micelles, it tends to that observed in the hydro-organic mode. The elution behaviour of the β-blockers was modelled to predict the retention times. This allowed the detailed exploration of the selectivity and resolution of the chromatographic systems in relatively wide ranges of concentration of surfactant and organic solvent. The best performance in terms of resolution and analysis time was achieved using HSC with acetonitrile, being able to base-line resolve a mixture of eight β-blockers. Ethanol also provided a good separation performance, significantly improved with respect to methanol and 1-propanol. In contrast, the hydro-organic mode using acetonitrile or any of the short-chain alcohols could not succeed with the separation of the β-blockers, owing to the poorer selectivity and wider peaks.     

Comparison of the performance of non-ionic and anionic surfactants as mobile phase additives in the RPLC analysis of basic drugs

Surfactants added to the mobile phases in reversed-phase liquid chromatography (RPLC) give rise to a modified stationary phase, due to the adsorption of surfactant monomers. Depending on the surfactant nature (ionic or non-ionic), the coated stationary phase can exhibit a positive net charge, or just change its polarity remaining neutral. Also, micelles in the mobile phase introduce new sites for solute interaction. This affects the chromatographic behavior, especially in the case of basic compounds. Two surfactants of different nature, the non-ionic Brij-35 and the anionic sodium dodecyl sulfate (SDS) added to water or aqueous-organic mixtures, are here compared in the separation of basic compounds (β-blockers and tricyclic antidepressants). The reversible/irreversible adsorption of the monomers of both surfactants on the stationary phase was examined. The changes in the nature of the chromatographic system using different columns and chromatographic conditions were followed based on the changes in retention and peak shape. The study revealed that Brij-35 is suitable for analyzing basic compounds of intermediate polarity, using "green chemistry", since the addition of an organic solvent is not needed and Brij-35 is a biodegradable surfactant. In contrast, RPLC with hydro-organic mixtures or mobile phases containing SDS required high concentrations of organic solvents.     

Submicellar and micellar reversed-phase liquid chromatographic modes applied to the separation of β-blockers

The behaviour of a reversed-phase liquid chromatographic (RPLC) system (i.e. elution order, resolution and analysis time), used in the analysis of β-blockers with acetonitrile–water mobile phases, changes drastically upon addition of an anionic surfactant (sodium dodecyl sulphate, SDS). Surfactant monomers cover the alkyl-bonded phase in different extent depending on the concentration of both modifiers, in the ranges 1 × 10⁻³–0.15 M SDS and 5–50% acetonitrile. Meanwhile, the surfactant is dissolved in the mobile phase as free monomers, associated in small clusters or forming micelles. Four characteristic RPLC modes are yielded, with transition regions between them: hydro-organic, micellar, and low and high submicellar. The mobile phases in the two latter modes contain a concentration of SDS below or well above the critical micellar concentration (CMC) in water (i.e. 8 × 10⁻³ M), and more than 30% acetonitrile. High submicellar RPLC appeared as the most promising mode, as it allowed full resolution of the β-blockers in practical times, while these were unresolved or highly retained in the other RPLC modes. The strong attraction of the cationic solutes to the anionic SDS makes a direct transfer mechanism between surfactant molecules in the stationary and mobile phases likely.     

Chromatographic Behavior of Aromatic Diamines in Hydro-Organic, Micellar and Submicellar Reversed Phase Liquid Chromatographic Modes

The chromatographic behavior (retention, elution strength, efficiency, peak asymmetry and selectivity) of some aromatic diamines in the presence of methanol with or without anionic surfactant SDS in the four different reversed phased liquid chromatographic (RPLC) modes, i.e., hydro-organic, micellar (MLC), low submicellar (LSC) and high submicellar (HSC), was investigated. In the three surfactant-mediated modes, the surfactant monomers coat the stationary phase even up to 70 % methanol; this results in the suppression of peak tailing (by masking the silanol groups on the stationary phase). In MLC and HSC, the solute retention decreases by increasing the surfactant concentration, while this situation was reversed in LSC. In the region between MLC and HSC modes (25–50 % methanol), retention of late eluting solutes was increased by increasing methanol content which is seemingly due to disaggregation of SDS micelles. Changes in selectivity were observed after changing the concentrations of SDS and methanol, in a greater extent when concentration of SDS was changed. Among the four studied RPLC modes, HSC showed the best efficiency with nearly symmetrical peaks. Prediction of retention of solutes in HSC based on a mechanistic retention model combined with Pareto-optimality method allowed the full resolution of target diamines in practical analysis times.     

Adsorption of the anionic surfactant sodium dodecyl sulfate on a C18 column under micellar and high submicellar conditions in reversed‐phase liquid chromatography

Micellar liquid chromatography makes use of aqueous solutions or aqueous‐organic solutions containing a surfactant, at a concentration above its critical micelle concentration. In the mobile phase, the surfactant monomers aggregate to form micelles, whereas on the surface of the nonpolar alkyl‐bonded stationary phases they are significantly adsorbed. If the mobile phase contains a high concentration of organic solvent, micelles break down, and the amount of surfactant adsorbed on the stationary phase is reduced, giving rise to another chromatographic mode named high submicellar liquid chromatography. The presence of a thinner coating of surfactant enhances the selectivity and peak shape, especially for basic compounds. However, the risk of full desorption of surfactant is the main limitation in the high submicellar mode. This study examines the adsorption of the anionic surfactant sodium dodecyl sulfate under micellar and high submicellar conditions on a C₁₈ column, applying two methods. One of them uses a refractive index detector to obtain direct measurements of the adsorbed amount of sodium dodecyl sulfate, whereas the second method is based on the retention and peak shape for a set of cationic basic compounds that indirectly reveal the presence of adsorbed monomers of surfactant on the stationary phase.     

Peak half-width plots to study the effect of organic solvents on the peak performance of basic drugs in micellar liquid chromatography

The addition of the anionic surfactant sodium dodecyl sulphate (SDS) to hydro-organic mixtures of methanol, ethanol, propanol or acetonitrile with water yielded enhanced peak shape (i.e. increased efficiencies and symmetrical peaks) for a group of basic drugs (β-blockers) chromatographed with a Kromasil C18 column. The effect can be explained by the thin layer of surfactant associated to the hydrocarbon chain on the stationary phase in the presence of the organic solvents, which covers the free silanols on the siliceous support avoiding their interaction with the cationic basic drugs. These instead interact with the anionic head of the surfactant increasing their retention and allowing a more facile mass transfer. The peak shape behaviour with the four organic solvents (methanol, ethanol, propanol and acetonitrile) was checked in the presence and absence of SDS. The changes in peak broadening rate and symmetry inside the chromatographic column were assessed through the construction of peak half-width plots (linear relationships between the left and right half-widths at 10% peak height versus the retention time). The examination of the behaviour for a wide range of compositions indicated that the effect of acetonitrile in the presence of SDS is different from ethanol and propanol, which behave similarly. Acetonitrile seems to be superior to the alcohols in terms of peak shape, which can be interpreted by the larger reduction in the adsorbed surfactant layer on the C18 column. However, the decreased efficiencies observed at increasing surfactant concentration in the mobile phase should be explained by the reduction in retention times, more than by a change in the stationary phase nature.     

Retention mechanisms in micellar liquid chromatography

Micellar liquid chromatography (MLC) is a reversed-phase liquid chromatographic (RPLC) mode with mobile phases containing a surfactant (ionic or non-ionic) above its critical micellar concentration (CMC). In these conditions, the stationary phase is modified with an approximately constant amount of surfactant monomers, and the solubilising capability of the mobile phase is altered by the presence of micelles, giving rise to diverse interactions (hydrophobic, ionic and steric) with major implications in retention and selectivity. From its beginnings in 1980, the technique has evolved up to becoming a real alternative in some instances (and a complement in others) to classical RPLC with hydro-organic mixtures, owing to its peculiar features and unique advantages. This review is aimed to describe the retention mechanisms (i.e. solute interactions with both stationary and mobile phases) in an MLC system, revealed in diverse reports where the retention behaviour of solutes of different nature (ionic or neutral exhibiting a wide range of polarities) has been studied in a variety of conditions (with ionic and non-ionic surfactants, added salt and organic solvent, and varying pH). The theory is supported by several mechanistic models that describe satisfactorily the retention behaviour, and allow the measurement of the strength of solute-stationary phase and solute-micelle interactions. Suppression of silanol activity, steric effects in the packing pores, anti-binding behaviour, retention of ionisable compounds, compensating effect on polarity differences among solutes, and the contribution of the solvation parameter model to elucidate the interactions in MLC, are commented.     

Enhancement of selectivity in reversed-phase liquid chromatography

In an effort to gain insight into the relationship between stationary phase solvation and selectivity, the use of short- and medium-chained-length alcohols (methanol, n-propanol, n-butanol, and n-pentanol) as mobile phase modifiers in reversed-phase liquid chromatography (RPLC) was investigated to determine their impact on chromatographic selectivity. A wide range of mobile phase compositions was evaluated because of the large effect exerted by solvent strength on selectivity. Employing a set of six vanillin compounds as retention probes, evidence is presented to support the view that an increase in the hydrophobicity of the organic modifier used in RPLC can increase the selectivity of the C18 alkyl bonded phase while simultaneously decreasing the retention time of the eluting solutes. Thus, we are presented with an interesting paradox: higher selectivity and shorter retention times, which can be attributed to changes in either solvent selectivity and/or stationary phase solvation by the organic modifier.     

Modification of the murakami retention model in reversed-phase high-performance liquid chromatography for micellar chromatographic separations

A retention model for micellar liquid chromatography was tested based on the data of separation of three benzodiazepins and six β-blockers. The model was obtained by analyzing changes in the microenvironment of a sorbate in transferring from the mobile to stationary phase. It can be used to describe the retention of benzodiazepins, which are neutral under the separation conditions, and the positively charged β-blockers. The calculated model coefficients are indicative of an increase in the number of 1-pentanol molecules and sodium dodecyl sulfate monomers in the microenvironment of the sorbates in transferring from the mobile to stationary phase. The solvation of the positive β-blockers by anionic surfactant monomers was higher than that of neutral benzodiazepins.     

醋酸水流动相体系中硅胶键合相上生物大分子保留行为的研究

 提出了以醋酸 水作为流动相的体系中 ,在ODS柱上分离生物大分子的反相高效液相色谱 (RPLC)方法。实验结果表明 ,醋酸 水的洗脱能力强于甲醇 水 三氟醋酸体系 ,在一定程度上克服了色谱分离中一些蛋白质的不可逆吸附且具有便于冷冻干燥的优点。用参数Z(1mol溶剂化溶质被溶剂化固定相吸附时从两者接触表面释放出置换剂的摩尔总数 ) ,logI(与 1mol溶质对固定相亲和势有关的常数 )和 j(与 1mol溶剂对固定相亲和势有关的常数 )对 9种蛋白质在此流动相体系中的保留进行了表征。     

Prediction of peak shape in hydro-organic and micellar-organic liquid chromatography as a function of mobile phase composition

A simple model is proposed that relates the parameters describing the peak width with the retention time, which can be easily predicted as a function of mobile phase composition. This allows the further prediction of peak shape with global errors below 5%, using a modified Gaussian model with a parabolic variance. The model is useful in the optimisation of chromatographic resolution to assess an eventual overlapping of close peaks. The dependence of peak shape with mobile phase composition was studied for mobile phases containing acetonitrile in the presence and absence of micellised surfactant (micellar-organic and hydro-organic reversed-phase liquid chromatography, RPLC). In micellar RPLC, both modifiers (surfactant and acetonitrile) were observed to decrease or improve the efficiencies in the same percentage, at least in the studied concentration ranges. The study also revealed that the problem of achieving smaller efficiencies in this chromatographic mode, compared to hydro-organic RPLC, is not only related to the presence of surfactant covering the stationary phase, but also to the smaller concentration of organic solvent in the mobile phase.     

原子转移自由基聚合制备新型接枝聚α-D-吡喃半乳糖苷色谱固定相及其性能

以2-溴异丁酰溴为引发剂,CuCl/CuC12/Me6TREN为催化体系,在室温条件下采用原子转移自由基聚合(ATRP)法将单体6-O-甲基丙烯酰基-1,2,3,4-双-O-亚异丙基-α-D-吡喃半乳糖苷(6-O-methacryloyl-1,2,3,4-di-O-isopropylidene-α-D-galactop...     

The role of the dual nature of ionic liquids in the reversed-phase liquid chromatographic separation of basic drugs

The cationic nature of basic drugs gives rise to broad asymmetrical chromatographic peaks with conventional C18 columns and hydro-organic mixtures, due to the ionic interaction of the positively charged solutes with the free silanol groups on the alkyl-bonded reversed-phase packing. Ionic liquids (ILs) have recently attracted some attention to reduce this undesirable silanol activity. ILs are dual modifiers (with a cationic and anionic character), which means that both cation and anion can be adsorbed on the stationary phase, giving rise to interesting interactions with the anionic free silanols and the cationic basic drugs. A comparative study of the performance of four imidazolium-based ILs as modifiers of the chromatographic behaviour of a group of β-blockers is shown. The ILs differed in the adsorption capability of the cation and anion on C18 columns. Mobile phases without additive and containing a cationic (triethylamine, TEA) or anionic (sodium dodecyl sulphate, SDS) additive were used as references for the interpretation of the behaviours. The changes in the nature of the chromatographic system, at increasing concentration of the additives, were followed based on the changes in retention and peak shape of the β-blockers. The silanol suppressing potency of the additives, and the association constants between the solutes and modified stationary phase or additive in the mobile phase, were estimated. The study revealed that SDS and the ionic liquid 1-hexyl-3-methylimidazolium tetrafluoroborate are the best enhancers of chromatographic peak shape among those studied.     

Use of a three-factor interpretive optimisation strategy in the development of an isocratic chromatographic procedure for the screening of diuretics in urine samples using micellar mobile phases

Screening of diuretics in urine is feasible through direct injection of the samples into the chromatographic system and isocratic reversed-phase liquid chromatography (RPLC) with micellar-organic mobile phases of sodium dodecyl sulfate (SDS) and 1-propanol. The surfactant coverage of the chromatographic column makes the addition of organic competing amines less necessary than in conventional aqueous-organic RPLC to achieve well-shaped peaks. Also, the range of elution strengths of micellar mobile phases required to elute mixtures of hydrophobic and hydrophilic diuretics is smaller. This allows the isocratic separation of the diuretics within adequate analysis times. An interpretive methodology is applied to optimise the resolution of a mixture of 15 diuretics of diverse polarity and acid-base behaviour (althiazide, amiloride, bendroflumethiazide, benzthiazide, bumetanide, canrenoic acid, chlorthalidone, ethacrynic acid, furosemide, piretanide, probenecid, torasemide, triamterene, trichloromethiazide and xipamide), using pH and concentrations of surfactant and organic modifier in the mobile phase as separation factors. Twelve diuretics were resolved in 25 min using 0.055 M SDS-6.0% 1-propanol at pH 3.0. The mixture of 15 diuretics was also resolved with two mobile phases showing complementary behaviour: 0.05 M SDS-5.6% 1-propanol at pH 5.4 and 0.11 M SDS-5.4% 1-propanol at pH 4.2. The results were applied to the analysis of urine samples with limits of detection similar to those usually reported for aqueous-organic RPLC, taking into account that the samples were injected without any previous treatment to separate or preconcentrate the analytes.     

Capillary electrophoretic studies on the migration behavior of cationic solutes and the influence of interactions of cationic solutes with sodium dodecyl sulfate on the formation of micelles and critical micelle concentration

The migration behavior of cationic solutes and influences of the interactions of cationic solutes with sodium dodecyl sulfate (SDS) on the formation of micelles and its critical micelle concentration (CMC) were investigated by capillary electrophoresis at neutral pH. Catecholamines and structurally related compounds, including epinephrine, norepinephrine, dopamine, norephedrine, and tyramine, which involve different extents of hydrophobic, ionic and hydrogen-bonding interactions with SDS surfactant, are selected as cationic solutes. The dependence of the effective electrophoretic mobility of cationic solutes on the concentration of surfactant monomers in the premicellar region provides direct evidence of the formation of ion-pairs between cationic solutes and anionic dodecyl sulfate monomers. Three different approaches, based on the variations of either the effective electrophoretic mobility or the retention factor as a function of surfactant concentration in the premicellar and micellar regions, and the linear relationship between the retention factor and the product of a distribution coefficient and the phase ratio, were considered to determine the CMC value of SDS micelles. The suitability of the methods used for the determination of the CMC of SDS with these cationic solutes was discussed. Depending on the structures of cationic solutes and electrophoretic conditions, the CMC value of SDS determined varies in a wide concentration range. The results indicate that, in addition to hydrophobic interaction, both ionic and hydrogen-bonding interactions have pronounced effects on the formation of SDS micelles. Ionic interaction between cationic solutes and SDS surfactant stabilizes the SDS micelles, whereas hydrogen-bonding interactions weakens the solubilization of the attractive ionic interaction. The elevation of the CMC of SDS depends heavily on hydrogen-bonding interactions between cationic solutes and SDS surfactant. Thus, the CMC value of SDS is remarkably elevated with catecholamines, such as epinephrine and norepinephrine, as compared with norephedrine. In addition, the effect of methanol content in the sample solution of these cationic solutes on the CMC of SDS was also examined.     

Characterization of C18-bonded liquid chromatographic stationary phases by Raman spectroscopy: the effect of mobile phase composition

Raman spectroscopy is used to examine the effect of mobile phase composition on the orientation of octadecyl-bonded silica-based reversed-phase liquid chromatographic (RPLC) stationary phase ligands. The effect of ligand bonding density is also investigated. The present experimental set-up utilizes a direct, noninvasive, on-column approach to examine the solvent dependent conformational behavior of the bonded ligands under flow-rate and back pressure conditions similar to those used during conventional RPLC measurements. Neat, single-component, mobile phase solvents including water, acetonitrile, methanol and chloroform are used to investigate the hypothesized collapsing and extension of stationary phase ligands with changes in mobile phase composition. No evidence of phase collapse was observed upon changing the mobile phase composition from an organic to an aqueous content. Also, Raman spectroscopic measurements allowed the differentiation between associated and free acetonitrile solvent.     

A novel approach to study the inclusion mechanism of imidazole derivatives in micellar chromatography

A chromatographic approach was proposed to describe the existence of surfactant micelles in a surfactant/hydroorganic phosphate buffer mobile phase. Using this mixture as a mobile phase, a novel mathematical theory is presented to describe the inclusion mechanism of imidazole derivatives in surfactant micelles. Using this model, enthalpy, entropy and the Gibbs free energy were determined for two chromatographic chemical processes: (i) the transfer of the imidazole derivative from the mobile phase to the stationary phase; and (ii) the imidazole derivative inclusion in surfactant micelles. These thermodynamic data indicate that the main parameter determining chromatographic retention is distribution of the imidazole derivatives to micelles of surfactant while the interaction with the stationary phase play a minor role.     

Development and evaluation of new zwitterionic hydrophilic interaction liquid chromatography stationary phases based on 3-P,P-diphenylphosphonium-propylsulfonate

New zwitterionic stationary phases were synthesized by covalently bonding 3-P,P-diphenylphosphonium-propylsulfonate to silica gel. The resulting materials possess both a negatively charged sulfonate group and a positively charged quaternary phosphonium group, which means that there is no net charge over a wide pH range. The retention mechanism and chromatographic behavior of polar solutes under HILIC conditions were studied on these zwitterionic phases. Compared to the commercial ZIC-HILIC column and a bare silica gel stationary phase, the newly synthesized zwitterionic stationary phases provided greater retention, higher peak efficiency and better peak symmetry in the HILIC mode. The analytes examined included: β-blockers, nucleic acid bases and nucleosides, salicylic acid and its analogues, and water soluble vitamins. Factors, such as the type of organic modifiers, solvent composition, pH and the buffer concentration of the mobile phase, have been considered as potential variables for controlling the chromatographic retention of polar analytes.     

An Integrated Theory of Adsorption and Partition Mechanism and Eash Contribution to Solute Retention in Reversed Phase Liquid Chromatography

With the combination of the the stoichiometric displacement model for retention (SDM-R) in reversed phase liquid chromatography (RPLC) and the stoichiometric displacement model for adsorption (SDM-A) in physical chemistry,the total number of moles of the re-solvated methanol of stationary phase side.nr,and that of solute side in the mobile phase,q,corresponding the one mole of the desorbing solute,were separately determined and referred as the characterization parameters of the contributions of the adsorption mechanism and partition mechanism to the solute retention,respectively.A chromatographic system of insulin,using mobile phase consisting of the pseudo-homologue of alcohols(methanol,ethanol and 2-propanol)-water and trifluoroacetic acid was employed.The maximum number of the methanol layers on the stationary phase surface was found to be 10.6,only 3 of which being valid in usual RPLC,traditionally referred as a volume process in partition mechanism.However,it still follows the SDM-R.Both of q and nr of insulin were found not to be zero,indicating that the retention mechanism of insulin is a mixed mode of partition mechanism and adsorption mechanism.When methanol is used as the organic modifier,the ratio of q/nr was 1.13,indicating the contribution to insulin retention due to partition mechanism being a bit greater than that due to adsorption mechanism.A linear relationship between q,or nr and the carbon number of the pseudo-homologue in the mobile phase was also found.As a methodology for investigating the retention mechanism retention and behavior of biopolymers.a homologue of organic solvents as the organic modifier in mobile phase has also been explored.     

Selectivity comparison of acetonitrile-methanol-water ternary mobile phases on an octadecylsiloxane-bonded silica stationary phase

The solvation parameter model was used in this study to investigate various intermolecular interactions that influence retention on the standard C18 stationary phase for the solvent system acetonitrile:methanol (ACN:MeOH, 1:1). In comparison to the organic mobile phase modifiers acetonitrile, acetone, methanol, 2-propanol, and tetrahydrofuran, the solvent strength for the ACN:MeOH (1:1) solvent system was evaluated. To facilitate the interpretation of various intermolecular interactions that contribute to retention on a standard C18 stationary phase for the solvent system ACN:MeOH (1:1), system maps were constructed and compared with those of acetone, tetrahydrofuran, acetonitrile, 2-propanol, and methanol. The solvation parameter models were constructed for the ternary solvent system ACN:MeOH (1:1)-water, and in the models constructed, the coefficient of determination values were from 0.998 to 0.999, the Fisher statistic values for the models were from 1687 to 4015, and the standard error of the estimate values ranged from 0.022 to 0.029. The solvent system ACN:MeOH (1:1) has retention properties more similar to methanol than acetonitrile, indicating methanol's influence is more dominant.