Improved efficiency in micellar liquid chromatography using triethylamine and 1-butanol as mobile phase additives to reduce surfactant adsorption

The effect of triethylamine as a mobile phase modifier on chromatographic efficiency in micellar liquid chromatography (MLC) is reported for nine different columns with various bonded stationary phases and silica pore sizes, including large-pore short alkyl chain, non-porous, and perfluorinated. Reduced plate height (h) versus reduced velocity (nu) plots were constructed for each column and the A' and C' terms calculated using a simplified Van Deemter equation introduced in our previous work. To further explore the practicality of using triethylamine in the micellar mobile phase, the efficiency of nine polar and non-polar substituted benzenes was studied on seven columns. Surfactant adsorption isotherms were measured for five columns with three micellar mobile phases to understand the relationship between adsorbed surfactant, mobile phase additive, and column efficiency. Clear improvements in efficiency were observed with the addition of 2% (v/v) triethylamine to a 1-butanol modified aqueous micellar mobile phase. This finding is supported by the lower amount of surfactant adsorbed onto the stationary phase when TEA is present in the mobile phase compared to an SDS only or a 1-butanol modified SDS mobile phase.     

Chromatographic analysis of phenethylamine-antihistamine combinations using C8, C18 or cyano columns and micellar sodium dodecyl sulfate-pentanol mixtures

The chromatographic behaviour of binary and ternary mixtures of several phenethylamines (phenylephrine, phenylpropanolamine, ephedrine, pseudoephedrine and methoxyphenamine) and antihistamines (pheniramine, carbinoxamine, doxylamine, chlorpheniramine, dexchlorpheniramine, dexbrompheniramine, diphenhydramine, tripolidine, azatadine and phenyltoloxamine), found in cough-cold pharmaceutical preparations, was studied using C8, C18 and cyano columns, micellar mobile phases of sodium dodecyl sulfate (SDS) and pentanol and UV detection. Using a C8 column and mobile phases of 0.05 mol l-1 SDS-6% v/v pentanol or 0.15 mol l-1 SDS-2% v/v pentanol at pH 7, more than 30 different phenethylamine-antihistamine combinations can be resolved in < 15 min. Intra- and inter-day repeatabilities and reproducibilities evaluated at three different drug concentrations (0.5, 5 and 25 micrograms ml-1, n = 10) were below 1.6, 2.5 and 2.4%, respectively. The drug amounts found in 18 formulations agreed with those declared by the manufacturers within the tolerance limits, and with those obtained using a mobile phase of 55% v/v methanol at pH 7. No interference was observed from other accompanying drugs such as acetylsalicylic acid, ascorbic acid, betamethasone, bromhexine, caffeine, codeine, dextromethorphan, paracetamol, prednisolone, salicylamide and tartrazine. The proposed procedure has the advantage over the conventional aqueous-organic procedure of using a small amount of organic solvent, which is highly retained in the SDS solution. The efficiencies are also greater. On the other hand, in the micellar system, the retentions of phenethylamines and antihistamines are similar, although the compounds can be easily resolved. In contrast, using the methanol-water mobile phase, the phenethylamines are weakly retained, whereas the antihistamines usually show a high retention.     

Comparison of the performance of butanol and pentanol as modifiers in the micellar chromatographic determination of some phenethylamines

A procedure was developed for the determination of several phenethylamines (amphetamine, arterenol, ephedrine, phenylephrine, phenylpropanolamine, mephentermine, methoxyphenamine, pseudoephedrine and tyramine), using micellar mobile phases of sodium dodecyl sulfate (SDS), a C18 column and UV detection. The drugs were eluted at short retention times with conventional acetonitrile-water or methanol-water mobile phases. In contrast, in the micellar system, they were strongly retained due to association with the surfactant adsorbed on the stationary phase, and needed the addition of butanol or pentanol to be eluted from the column. These modifiers allowed a simple way of controlling the retention. The chromatographic efficiencies obtained with the hybrid mobile phases of SDS-butanol and SDS-pentanol were also very high, mostly in the N=3000-7000 range, significantly greater than those achieved with a conventional acetonitrile-methanol-water mobile phase. Butanol and pentanol yielded similar selectivities, but the latter modifier permitted significantly shorter retention times than butanol, and was preferred to expedite the analysis of the pharmaceuticals. Most binary combinations of the nine phenethylamines can be resolved with these mobile phases. A mobile phase of 0.15 M SDS-5% pentanol was used to assay five of the phenethylamines (amphetamine, ephedrine, phenylephrine, phenylpropanolamine and pseudoephedrine) in 22 pharmaceutical preparations, which contained diverse accompanying compounds. The results agreed with the declared compositions and with those obtained with a mobile phase of methanol-acetonitrile-0.05 M phosphate buffer (pH 3) 10:5:85, with no interferences and relative errors usually below 2%. However, with the aqueous-organic mobile phase, the retention time for phenylephrine was too low and could not be usually evaluated.     

Determination of active ingredients in cough-cold preparations by micellar liquid chromatography

The chromatographic behaviour of some active ingredients in cough-cold pharmaceutical preparations, the antihistamine chlorpheniramine (or the dextro enantiomer dexchlorpheniramine), and the phenethylamines phenylephrine, phenylpropanolamine and pseudoephedrine, has been studied using a C(18) column, micellar mobile phases of sodium dodecyl sulphate (SDS) and pentanol, and with UV detection. All possible combinations of chlorpheniramine/phenethylamine were resolved and determined using a mobile phase of 0.15 M SDS-6% (v/v) pentanol at pH 7, with analysis time below 7 min. Repeatabilities and within laboratory precisions were evaluated at four different drug concentrations in the range 0.5-25 mug ml(-1) (n=5), resulting RSDs below 1.6%. The drug amounts found in the analysis of 14 commercialised preparations agreed with those declared by the manufacturers within the tolerance limits, and with those obtained using an aqueous 60% (v/v) methanol reference mobile phase. No interference was observed from other accompanying drugs such as acetylsalicylic acid, ascorbic acid, betamethasone, caffeine, codeine phosphate, diphenhydramine, lactose, paracetamol, and prednisolone. The studied combinations required a rather high amount of methanol in conventional RPLC to be eluted from the column. In contrast, the proposed procedure used a much lower amount of organic solvent (pentanol), which is highly retained in the SDS solution, being also less toxic than methanol.     

Therapeutic monitoring of imipramine and desipramine by micellar liquid chromatography with direct injection and electrochemical detection

A micellar liquid chromatographic (MLC) procedure was developed for the clinical monitoring of imipramine and its active metabolite, desipramine. The determination of these highly hydrophobic substances was carried out after direct injection of the serum samples using a mobile phase composed of 0.15 m SDS--6% (v/v) pentanol buffered at pH 7, pumped at 1.5 mL/min into a C(18) column (250 x 4.6 mm), and electrochemical detection at 650 mV. Using this MLC method, calibration was linear (r > 0.995) and the limits of detection (ng/mL) were 0.34 and 0.24 for imipramine and desipramine, respectively. Repeatabilities and intermediate precision were tested at three different concentrations in the calibration range and a CV (%) below 2.2 was obtained. In this MLC procedure, the serum is determined without treatment, thus allowing repeated serial injections without changes in retention factors, and reducing the time and consumables required to carry out the pretreatment process. The assay method can be applied to the routine determination of serum imipramine and its metabolite in therapeutic drug monitoring.     

Capillary liquid chromatography as a tool for separation of hydrophobic basic drugs. Relation between tests for column characterization and real analysis

Two capillary columns for reversed phase (RP) capillary liquid chromatography (CLC), viz. Nucleosil 100‐5 C18 and LiChrosorb RP‐select B, were characterized by the Walters test, i.e. the chromatographic test proposed for RP stationary phases. Hydrophobicity indices were determined not only in acetonitrile/water mobile phase, as proposed in the test, but they were also measured in buffered systems. This approach was used to quantify the influence of mobile phase composition on the modification of the surface of the stationary phases. In the next step, small basic compounds differing in their hydrophobicity and basicity were selected and their retention on the stationary phases in mobile phases of the same composition as used for column testing was examined. Furthermore, the retention of newly synthesized drugs, chemotherapeutics derived from thioacridine and pyridoquinoline, differing in their structures, basicity, and hydrophobicity, was also studied. The composition of the mobile phases had to be shifted to higher contents of organic modifiers – acetonitrile or methanol – in order to elute these hydrophobic compounds from the columns. The question we wanted to answer was: How is the method for testing of reversed phases related to retention, separation efficiency, and peak symmetry of various analytes?     

Efficiency enhancements in micellar liquid chromatography through selection of stationary phase and alcohol modifier

Micellar liquid chromatography (MLC) remains hindered by reduced chromatographic efficiency compared to reversed phase liquid chromatography (RPLC) using hydro-organic mobile phases. The reduced efficiency has been partially explained by the adsorption of surfactant monomers onto the stationary phase, resulting in a slow mass transfer of the analyte within the interfacial region of the mobile phase and stationary phase. Using an array of 12 columns, the effects of various bonded stationary phases and silica pore sizes, including large-pore short alkyl chain, non-porous, superficially porous and perfluorinated, were evaluated to determine their impact on efficiency in MLC. Additionally, each stationary phase was evaluated using 1-propanol and 1-butanol as separate micellar mobile phase alcohol additives, with several columns also evaluated using 1-pentanol. A simplified equation for calculation of A' and C' terms from reduced plate height (h) versus reduced velocity (nu) plots was used to compare the efficiency data obtained with the different columns and mobile phases. Analyte diffusion coefficients needed for the h versus nu plots were determined by the Taylor-Aris dispersion technique. The use of a short alkyl chain, wide-pore silica column, specifically, Nucleosil C4, 1000A, was shown to have the most improved efficiency when using a micellar mobile phase compared to a hydro-organic mobile phase for all columns evaluated. The use of 1-propanol was also shown to provide improved efficiency over 1-butanol or 1-pentanol in most cases. In a second series of experiments, column temperatures were varied from 40 to 70 degrees C to determine the effect of temperature on efficiency for a subset of the stationary phases. Efficiency improvements ranging from 9% for a Chromegabond C8 column to 58% for a Zorbax ODS column were observed over the temperature range. Based on these observed improvements, higher column temperatures may often yield significant gains in column efficiency, assuming the column is thermally stable.     

Aliphatic carboxylic acids and alcohols as efficiency and elution strength enhancers in micellar liquid chromatography

Micellar liquid chromatography (MLC) uses surfactant solutions as mobile phases with added organic additives to enhance both the elution strength and the chromatographic efficiency. Two aliphatic carboxylic acids (1-butanoic and 1-pentanoic) were used as MLC additives and compared with the two corresponding alcohols (1-butanol, 1-pentanol) in terms of elution strength, efficiency and selectivity. A set of 11 phenol derivatives was used as probe compounds. All micellar mobile phases were prepared with sodium dodecylsulfate (SDS) with concentration ranging from 0.05 to 0.15 M and the modifier content within 1.0 and 5.0% (v/v). The elution strength of different mobile phases containing a constant amount of SDS and different amounts of modifiers; and mobile phases containing a constant amount of modifier and different SDS concentration were determined and discussed. The effect of the acid modifiers on efficiency was studied constructing van Deemter plots that showed no minimum within the 0.01–0.7 mL/min flow rate range studied. Temperature effects were also studied constructing the classical van’t Hoff plots. The slight curvature of the plots in the 25–70 °C range may indicate some modification of the surfactant-bonded moiety layer on the stationary phase surface. Since no definitive advantage of the use of aliphatic acids were established compared to their alcohol counterpart, their terrible smell will probably preclude their use as MLC organic modifiers.     

Micellar Liquid Chromatography Study of Quantitative Retention–Activity Relationships for Antihypertensive Drugs

Use of micellar mobile phases in reversed-phase liquid chromatography (RPLC) results in hydrophobic and electrostatic sites for interaction. Modified stationary phases in micellar liquid chromatography (MLC) are structurally similar to biomembranes. To confirm this we focused on the effects of the type and concentration of surfactant (Brij 35, SDS, and CTAB) and mobile phase pH on the retention of antihypertensive drugs on modified C₁₈ stationary phases. Quantitative retention-activity relationships are proposed for the drugs and the different surfactants and compared with those obtained using aqueous–organic mobile phases. Finally, a correlation was obtained between the logarithm of retention factors (log k) and the toxicity (LD₅₀) of antihypertensive drugs. Revised: 14 September 2005 and 4 April 2006     

阴离子交换整体柱对蛋白质的分离与纯化

分别用乙二胺、二乙胺、三乙胺将自制的以甲基丙烯酸缩水甘油酯(GMA)为单体、乙二醇二甲基丙烯酸酯(EDMA)为交联剂的整体柱修饰为弱、强阴离子交换整体柱。考察了该整体柱的性能,选择出分离蛋白质(牛血清白蛋白、溶菌酶和谷胱甘肽)的最佳实验条件,并在最佳分离条件下考察了这些蛋白质在整体柱上的色谱行为和该整体柱对纤维素降解酶的分离纯化情况。实验结果表明,该整体柱性能良好,可以实现对纤维素降解酶的快速分离与纯化。同时,实验也证明采用梯度洗脱可以实现对某些蛋白质的分离纯化。     

Temperature and pH‐stability of commercial stationary phases

In this paper, the temperature and pH stability of silica‐based RP stationary phases were investigated. Furthermore, nonsiliceous phases like a polymeric column based on polystyrene divinylbenzene and a polybutadiene coated zirconium dioxide column were also included. The columns were heated up to 150°C at dynamic conditions, which means that the eluent consisting of water and methanol (90:10, v/v) was continuously purged through the packed bed. After every 5 h, the columns were cooled down to room temperature and the efficiency was measured by injecting a test sample based on the Neue test. It could be shown that some stationary phases exhibited a very good temperature stability at the test conditions specified above.     

How to select equivalent and complimentary reversed phase liquid chromatography columns from column characterization databases

Three RP-LC column characterization protocols [Tanaka et al. (1989), Snyder et al. (PQRI, 2002), and NIST SRM 870 (2000)] were evaluated using both Euclidian distance and Principal Components Analysis to evaluate effectiveness at identifying equivalent columns. These databases utilize specific chromatographic properties such as hydrophobicity, hydrogen bonding, shape/steric selectivity, and ion exchange capacity of stationary phases. The chromatographic parameters of each test were shown to be uncorrelated. Despite this, the three protocols were equally successful in identifying similar and/or dissimilar stationary phases.     

Micellar liquid chromatography: suitable technique for screening analysis

The screening capability of micellar liquid chromatography (MLC) is discussed using the reported chromatographic data of several sets of compounds (amino acids, beta-blockers, diuretics, phenethylamines, phenols, polynuclear aromatic hydrocarbons, steroids and sulfonamides) and new results (sulfonamides and steroids). The chromatographic data are treated with an interpretive optimisation resolution procedure to obtain the best separation conditions. Usually, the pH and the concentration of surfactant (sodium dodecyl sulfate, SDS, or cetyltrimethylammonium bromide) for the optimal mobile phase were 2.5-3 and < 0.12 M, respectively. The nature and concentration of organic solvent depended on the polarity of the eluted compounds: a low volume fraction of propanol (approximately 1%, v/v) was useful to separate the amino acids, with log P(o/w) < -1 (where P(o/w) is the octanol-water partition coefficient). A greater concentration of this solvent (approximately 5-7%) was needed for compounds in the range -1 < log P(o/w) < 2, as with the studied diuretics and sulfonamides, and a high concentration of propanol (approximately 15%) or a low concentration of butanol (< 10%) had to be used for less polar compounds with 1 < log P(o/w) < 3, such as the beta-blockers. Pentanol (< 6%) was more suitable for the even less polar compounds with log P(o/w) > 3, such as the steroids. For basic drugs such as the phenethylamines (0 < log P(o/w) < 1.7), eluted with a micellar eluent of anionic SDS, propanol was too weak. A study is also shown for mixtures of sulfonamides (log P(o/w) = -1.2 to 1.7) and steroids (log P(o/w) = 3.0-8.1) eluted from conventional C18 columns with SDS mobile phases containing acetonitrile and 1-pentanol, respectively, which are compared with classical acetonitrile-water and methanol-water mixtures. The results complement a previous study on beta-blockers (log P(o/w) = -0.03 to 2.8) and reveal that MLC is a very competitive technique for the screening of compounds against conventional RPLC, due to its peculiar behaviour with regard to the selectivity and elution strength. The concentration of organic solvent needed to obtain sufficiently low retention times (even for highly hydrophobic steroids with log P(o/w) = 7-8) is also appreciably smaller for MLC, which reduces the environmental impact of the mobile phases.     

Polycyclic aromatic hydrocarbons as test probes to investigate the retention behavior of 1,3‐alternate calix[4]arene silica‐bonded stationary phases

A series of polycyclic aromatic hydrocarbons (PAHs) of different size and shape has been used to characterize the chromatographic behavior of five calix[4]arene stationary phases in 1,3‐alternate conformation synthesized in our laboratory. The selection of linear, four‐ring nonlinear, and five‐ring PAHs gave data on selectivity changes across range of the calix[4]arene columns. Retention of the 12 aromatic solutes has been evaluated at various methanol contents in the mobile phase (70–100% v/v) and column temperatures (20–45°C). The thermodynamic parameters underlying the retention mechanisms revealed that each of the five calix[4]arene columns exhibited variation in selectivity and retention of PAHs caused by enthalpy and entropy effects. The calixarene stationary phases substituted with electron‐withdrawing groups exhibit enhanced selectivity toward PAHs in comparison to the rest of the investigated columns. The observed divergences are due to differences in solute–stationary phase interactions and originate in π–π and π‐electron transfer specific to the analytes and the type of calix[4]arene functionalization at the upper rim, as well as steric and sorption phenomena.     

Preparation and evaluation of diblock copolymer‐grafted silica by sequential surface initiated‐atom transfer radical polymerization for reverse‐phase/ion‐exchange mixed‐mode chromatography

A novel approach that involved the grafting of diblock copolymer with two types of monomer onto substrate by sequential surface initiated‐atom transfer radical polymerization was proposed to prepare a mixed‐mode chromatographic stationary phase. The distinguishing feature of this method is that it can be applied in the preparation of various mixed‐mode stationary phases. In this study, a new reverse‐phase/ion‐exchange stationary phase was prepared by grafting hydrophobic styrene and cationic sodium 4‐styrenesulfonate by the proposed approach onto silica surface. The chromatographic properties of the prepared stationary phase were evaluated by the separation of benzene derivatives, anilines, and β‐agonists, and by the effect of pH values and acetonitrile content on the retention. Compared with typical RP columns, the prepared stationary phase achieved the better resolution and higher selectivity at a shorter separation time and lower organic content. Moreover, the application of the prepared column was proved by separating widely distributed polar and charged compounds simultaneously.     

Investigation of polar stationary phases for the separation of sympathomimetic drugs with nano-liquid chromatography in hydrophilic interaction liquid chromatography mode

In this study, the retention and selectivity of a mixture of basic polar drugs were investigated in hydrophilic interaction chromatographic conditions (HILIC) using nano-liquid chromatography (nano-LC). Six sympathomimetic drugs including ephedrine, norephedrine, synephrine, epinephrine, norepinephrine and norphenylephrine were separated by changing experimental parameters such as stationary phase, acetonitrile (ACN) content, buffer pH and concentration, column temperature. Four polar stationary phases (i.e. cyano-, diol-, aminopropyl-silica and Luna HILIC, a cross-linked diol phase) were selected and packed into fused silica capillary columns of 100 μm internal diameter (i.d.). Among the four stationary phases investigated a complete separation of the all studied compounds was achieved with aminopropyl silica and Luna HILIC stationary phases only. Best chromatographic results were obtained employing a mobile phase composed by ACN/water (92/8, v/v) containing 10 mM ammonium formate buffer pH 3. The influence of the capillary temperature on the resolution of the polar basic drugs was investigated in the range between 10 and 50 °C. Linear correlation of ln k vs. 1/T was observed for all the columns; ΔH° values were negative with Luna HILIC and positive with aminopropyl- and diol-silica stationary phases, demonstrating that different mechanisms were involved in the separation.To compare the chromatographic performance of the different columns, Van Deemter curves were also investigated.     

Simultaneous determination of three opiates in serum by micellar liquid chromatography using direct injection

A simple and reliable micellar liquid chromatographic method was developed for the simultaneous determination of 3 opiates (codeine, morphine, and thebaine) in serum, using direct injection and ultraviolet detection. The separation of the drugs was optimized on a C18 column, thermostatically controlled at 25 degrees C, by evaluating mobile phases containing sodium dodecyl sulfate (SDS) and various modifiers (propanol, butanol, or pentanol). Adequate resolution of the opiates was obtained with a chemometrics approach, in which retention was modeled as a first step by using the retention factors for several mobile phases. Next, an optimization criterion that takes into account the position and shape of the chromatographic peaks was applied. The 3 opiates were totally resolved and determined in 12 min with the mobile phase 0.15M SDS-7% (v/v) butanol buffered at pH 7. The limits of detection for codeine and morphine were greatly improved by using fluorimetric detection. Repeatability and intermediate precision were tested for 3 different concentrations of the drugs, and the relative standard deviations were <0.8% for most of the assays. Finally, the method was successfully applied to the determination of morphine and codeine in serum samples.