Effect of buffer nature and concentration on the chromatographic performance of basic compounds in the absence and presence of 1-hexyl-3-methylimidazolium chloride.

In reversed-phase liquid chromatography, the performance for basic compounds is affected by the interaction of the protonated (cationic) species with the anionic free silanols on the alkyl-bonded stationary phases. Using aqueous-organic mobile phases in the absence of additives, the retention may be too high, and the peaks be broad and asymmetric. The performance is improved by addition to the mobile phase of ionic liquids, from which 1-hexyl-3-methylimidazolium chloride ([C6MIm][Cl]) has especially good characteristics. A recent report has also revealed that the use of the phosphate system as buffer, at varying concentration and pH, may have a significant role in the chromatographic performance of basic compounds, with effects on both retention and peak shape. In this work, this study has been extended to other three buffer systems (acetate, citrate, and formate), at increasing concentrations and pH 3 and 7, in the presence and absence of [C6MIm][Cl]. The results have been compared with those obtained with the phosphate system. The retention increases by addition of larger concentration of all buffers, in both absence and presence of [C6MIm][Cl]. Without additive, peak performance is also enhanced significantly. This effect is minimal in the presence of [C6MIm][Cl], which yields highly symmetrical peaks at all buffer concentrations, due to an effective blocking of the silanol activity.

Modulation of retention and selectivity in oil-in-water microemulsion liquid chromatography: A review.

Microemulsions (MEs) are stable, isotropically clear solutions consisting of an oil and water stabilized by a surfactant and a co-surfactant. Oil-in-water microemuslion liquid chromatography (MELC) is a relatively new chromatographic mode, which uses an O/W ME as mobile phase. Retention, selectivity and efficiency can be modified by changing the concentration of the ME components and the ratio between the aqueous and oil phases. This work makes a critical survey on the information found in the literature about the mobile phase compositions that lead to the creation of successful O/W ME mobile phases, as well as the effect of pH for ionizable compounds and temperature. The viability of performing the analyses using isocratic and gradient elution is also considered. The complexity of the composition of a successful ME, and the fact that the different factors interact each other, may require many manipulations during method development to achieve an acceptable separation for complex mixtures. This is the reason of the proposal from several authors of a standard ME as starting point when developing a method for a new separation with no previous reports. Based on these initial conditions, the interest of several authors in applying computer-assisted approaches to optimize the composition of ME mobile phases, and reduce significantly the time and reagent consumption for method development, is described. Some practical tips are given to prepare stable ME mobile phases that yield reproducible results.

Recent advances on ionic liquid uses in separation techniques.

The molten organic salts with melting point below 100°C, commonly called ionic liquids (ILs) have found numerous uses in separation sciences due to their exceptional properties as non molecular solvents, namely, a negligible vapor pressure, a high thermal stability, and unique solvating properties due to polarity and their ionic character of molten salts. Other properties, such as viscosity, boiling point, water solubility, and electrochemical window, are adjustable playing with which anion is associated with which cation. This review focuses on recent development of the uses of ILs in separation techniques actualizing our 2008 article (same authors, J. Chromatogr. A, 1184 (2008) 6-18) focusing on alkyl methylimidazolium salts. These developments include the use of ILs in nuclear waste reprocessing, highly thermally stable ILs that allowed for the introduction of polar gas chromatography capillary columns able to work at temperature never seen before (passing 300°C), the use of ILs in liquid chromatography and capillary electrophoresis, and the introduction of tailor-made ILs for mass spectrometry detection of trace anions at the few femtogram level. The recently introduced deep eutectic solvents are not exactly ILs, they are related enough so that their properties and uses in countercurrent chromatography are presented.

Extent of the influence of phosphate buffer and ionic liquids on the reduction of the silanol effect in a C18 stationary phase.

The presence of anionic free silanols in the silica-based stationary phases gives rise to broad and asymmetrical peaks when cationic basic compounds are chromatographed using hydro-organic mobile phases. The addition to the mobile phase of a reagent with ionic character prevents the access of analytes to the free silanols, improving the peak shape. The silanol activity can be affected by the buffer concentration and mobile phase pH, factors that are not always considered sufficiently in the literature. In this work, the chromatographic behaviour of three basic ß-adrenoceptor antagonists (acebutolol, nadolol and timolol), using mobile phases containing acetonitrile, was examined at different phosphate buffer concentrations (5-50mM) and mobile phase pH (2-8), in the absence and presence of three imidazolium-based ionic liquids (1-ethyl-, 1-butyl- and 1-hexyl-3-methylimidazolium chloride). All factors were evaluated through both the retention and peak shape. The imidazolium cations can block the access of cationic analytes through electrostatic interaction with the anionic silanols, or association with the alkyl chains bound to the stationary phase. In previous reports, the protection mechanism was demonstrated to be directly related to the cation size. The studies in this work reveal that the effectiveness of the mobile phase additive as silanol blocker also depends on the concentration of the buffer anion and the protonation degree of the silanols on the stationary phase. Increasing amounts of phosphate at low pH give rise to increasing retention times. Also, the peak shape is improved, which indicates the influence of phosphate on blocking the activity of free silanols. However, the benefits obtained by the combined effect of buffering the mobile phase at low pH and the use of a bulky additive are lost at pH>6.

Use of micellar liquid chromatography to analyze oxolinic acid, flumequine, marbofloxacin and enrofloxacin in honey and validation according to the 2002/657/EC decision.

A micellar liquid chromatographic method was developed for the analysis of oxolinic acid, flumequine, marbofloxacin and enrofloxacin in honey. These quinolines are unethically used in beekeeping, and a zero-tolerance policy to antibiotic residues in honey has been stated by the European Union. The sample pretreatment was a 1:1 dilution with a 0.05M SDS at pH 3 solution, filtration and direct injection, thus avoiding extraction steps. The quinolones were eluted without interferences using mobile phase of 0.05M SDS/12.5% 1-propanol/0.5% triethylamine at pH 3, running at 1mL/min under isocratic room through a C18 column. The analytes were detected by fluorescence. The method was successfully validated according to the requirements of the European Union Decision 2002/657/EC in terms of: specificity, linearity (r(2)>0.995), limit of detection and decision limit (0.008-0.070mg/kg), lower limit of quantification (0.02-0.2mg/kg), detection capability (0.010-0.10mg/kg), recovery (82.1-110.0%), precision (<9.4%), matrix effects, robustness (<10.4%), and stability. The procedure was applied to several commercial honey supplied by a local supermarket, and the studied antibiotics were not detected. Therefore, the method was rapid, simple, safe, eco friendly, reliable and useful for the routine analysis of honey samples.

On the use of ionic liquids as mobile phase additives in high-performance liquid chromatography. A review.

The popularity of ionic liquids (ILs) has grown during the last decades in several analytical separation techniques. Consequently, the number of reports devoted to the applications of ILs is still increasing. This review is focused on the use of ILs (mainly imidazolium-based associated to chloride and tetrafluoroborate) as mobile phase additives in high-performance liquid chromatography (HPLC). In this approach, ILs just function as salts, but keep several kinds of intermolecular interactions, which are useful for chromatographic separations. Both cation and anion can be adsorbed on the stationary phase, creating a bilayer. This gives rise to hydrophobic, electrostatic and other specific interactions with the stationary phase and solutes, which modify the retention behaviour and peak shape. This review updates the advances in this field, with emphasis on topics not always deeply considered in the literature, such as the mechanisms of retention, the estimation of the suppressing potency of silanols, modelling and optimisation of the chromatographic performance, and the comparison with other additives traditionally used to avoid the silanol problem.

Are analysts doing method validation in liquid chromatography?

Method validation is being applied in the reported analytical methods for decades. Even before this protocol was defined, authors already somehow validated their methods without full awareness. They wished to assure the quality of their work. Validation is an applied approach to verify that a method is suitable and rugged enough to function as a quality control tool in different locations and times. The performance parameters and statistical protocols followed throughout a validation study vary with the source of guidelines. Before single laboratory validation, an analytical method should be fully developed and optimized. The purpose of the validation is to confirm performance parameters that are determined during method development, and it should provide information on how the method will perform under routine use. An unstable method may require re-validation. Further method development and optimization will be needed if validation results do not meet the accepted performance standards. When possible, the validation protocol should also be conducted as a collaborative study by multiple laboratories, on different instruments, reagents, and standards. At this point, it would be interesting to know how people are validating their methods. Are they evaluating all defined validation parameters? Are they indicating the followed guidelines? Is re-validation really currently used? Is validation performed by a single laboratory, or is it a collaborative work by several laboratories? Is it an evolving discipline? In this survey, we will try to answer these questions focused to the field of liquid chromatography.

Performance of different C18 columns in reversed-phase liquid chromatography with hydro-organic and micellar-organic mobile phases.

Column selection in reversed-phase liquid chromatography (RPLC) can become a challenge if the target compounds interact with the silica-based packing. One of such interactions is the attraction of cationic solutes to the free silanols in silica-based columns, which is a slow sorption-desorption interaction process that gives rise to tailed and broad peaks. The effect of silanols is minimised by the addition of a competing agent in the mobile phase, such as the anionic surfactant sodium dodecyl sulphate (SDS). In micellar-organic RPLC, the adsorption of an approximately fixed amount of SDS monomers gives rise to a stable modified stationary phase, with properties remarkably different from those of the underlying bonded phase. The chromatographic behaviour (in terms of selectivity, analysis time and peak shape) of eight C18 columns in the analysis of weakly acidic phenols and basic ß-blockers was examined with hydro-organic and micellar-organic mobile phases. The behaviour of the columns differed significantly when the cationic basic drugs were eluted with hydro-organic mobile phases. With micellar-organic mobile phases, the adsorption of surfactant, instead of making the columns similar, gave rise to a greater diversity of behaviours (especially in terms of selectivity and analysis time), for both groups of phenols and ß-blockers, which should be explained by the residual effect of the underlying bonded stationary phase and the different amount of surfactant covering the packing. Therefore, the implementation of a micellar-organic procedure in RPLC will depend significantly on the selected type of C18 column.

Half-width plots, a simple tool to predict peak shape, reveal column kinetics and characterise chromatographic columns in liquid chromatography: state of the art and new results.

Peak profiles in chromatography are characterised by their height, position, width and asymmetry; the two latter depend on the values of the left and right peak half-widths. Simple correlations have been found between the peak half-widths and the retention times. The representation of such correlations has been called half-width plots. For isocratic elution, the plots are parabolic, although often, the parabolas can be approximated to straight-lines. The plots can be obtained with the half-widths/retention time data for a set of solutes experiencing the same kinetics, eluted with a mobile phase at fixed or varying composition. When the analysed solutes experience different resistance to mass transfer, the plots will be solute dependent, and should be obtained with the data for each solute eluted with mobile phases at varying composition. The half-width plots approach is a simple tool that facilitates the prediction of peak shape (width and asymmetry) with optimisation purposes, reveal the interaction kinetics of solutes in different columns, and characterise chromatographic columns. This work shows half-width plots for different situations in isocratic elution, including the use of different flows, the effect of temperature, the modification of the stationary phase surface by an additive, the existence of specific interactions within the column, and the comparison of columns. The adaptation to gradient elution is also described. Previous knowledge on half-width plots is structured and analysed, to which new results are added.

Validation of micellar LC-based methods applied to analyze foodstuffs.

The validation of several micellar LC-based analytical methodologies was described. These methods were able to quantify quinolones in fish from fisheries, hydroxytyrosol in olive extracts and biogenic amines in anchovy sauce. The validation was performed following the requirements of official guides to provide more reliability. Two guides suggested by renowned institution are described: US FDA Guidance for Industry and EU Regulation 2002/657/EC Decision. The appropriate guide was used for each method, depending of the analyte, the matrix and the scope of sample. The calculated validation parameters were those proposed by the guide: selectivity, calibration range, linearity, LOD and LOQ, inter- and intra-day accuracy and precision, limit of decision, detection capability, robustness, recovery and stability. The methodologies were successfully validated by the selected guideline, indicating their suitability to be applied to analysis of real samples, proven to be useful to its intended purpose.

Monitoring of HAART regime antiretrovirals in serum of acquired immunodeficiency syndrome patients by micellar liquid chromatography.

A methodology based on micellar liquid chromatography to monitor five antiretroviral drugs (lamivudine, stavudine, tenofovir, zidovudine and efavirenz) was proposed. Antiretrovirals were studied in sets of three, corresponding to each highly active antiretroviral therapy (HAART) regime, prescribed to acquired immunodeficiency syndrome (AIDS)-infected patients. Four aqueous micellar mobile phases buffered at pH 7 were optimized to separate these compounds, using sodium dodecyl sulfate as the tensioactive, and 1-propanol or 1-pentanol as the organic modifier. The composition of each mobile phase was optimized for each antiretroviral. The common separation conditions were: C18 apolar column (125 × 4.6 mm, 5 µm particle size), UV detection set at 214 nm, and mobile phase running at 1 mL min(-1) without controlling the temperature. The finally suggested method was validated for five analysed antiretroviral drugs following the US Food and Drug Administration guidelines in terms of: linearity between 0.5 and 50 ppm (r(2) > 0.9995), sensitivity (LOD lower than 0.25 ppm), intra- and inter-day precision (<7.1 and <5.2%, respectively) and accuracy (recovery 88.5-105.3% and 93.5-101.3%, respectively), as well as robustness (<6.5%). The proposed method was used to monitor the level of antiretrovirals in the serum of AIDS patients. The suggested methodology was found to be useful in the routine analysis of antiretrovirals in serum samples.

Hydrophobicity of ionisable compounds studied by countercurrent chromatography.

Countercurrent chromatography (CCC) is a liquid chromatography technique in which the stationary phase is also a liquid. The main chemical process involved in solute separation is partitioning between the two immiscible liquid phases: the mobile phase and the support-free liquid stationary phase. The octanol-water partition coefficients (P(o/w)) is the accepted parameter measuring the hydrophobicity of molecules. It is considered to estimate active principle partitioning over a biomembrane. It was related to the substance biological activity. CCC is able to work with an octanol stationary phase and an aqueous mobile phase. In this configuration, CCC is a useful and easy alternative to measure directly the P(o/w) of the molecules compared to other methods including the classical and tedious shake-flask method. Three ketones are used as model compounds to illustrate the CCC protocol of P(o/w) measurement. The focus of this work is put on ionisable molecules whose apparent P(o/w) is completely changed by ionization. ß-Blockers, diuretics and sulfonamides are compound classes that were studied. Some of the experimentally determined P(o/w) coefficients of the molecular forms disagreed with calculated and experimental values available in the literature. The P(o/w) coefficients of the ionic forms and the acidity constants were also calculated using a theoretical model. Relationships between biological properties and hydrophobicity are also discussed.

Analytical determination of hydroxytyrosol in olive extract samples by micellar liquid chromatography.

Hydroxytyrosol is a well-known natural phenolic component obtained from olive extract samples with antioxidant effects. A micellar liquid chromatography method to detect and quantify hydroxytyrosol in olive extract samples is described. Matrix resolution was performed using a Kromasil C18 column and a micellar mobile phase of sodium dodecyl sulphate (SDS) 0.05M and 4% methanol buffered at pH 7. Detection was set by absorbance at 280nm. Samples were diluted with 0.05M SDS at pH 3 and directly injected, thus avoiding long tedious extractions. Hydroxytyrosol was eluted in 3.5min without overlapping other matrix compounds. Validation was performed following the US FDA guideline. The main analytical parameters studied were: linearity (0.03-250µgmL-1; r2=0.999), limit of detection and quantification (3 and 30ngmL-1, respectively), intra- and inter-day precision (RSD, % <1.4 and <8.2, respectively), and robustness (RSD, %<6.6). Recoveries were in the 88.5-98.9% range.

Quinolones control in milk and eggs samples by liquid chromatography using a surfactant-mediated mobile phase.

Four quinolones (danofloxacin, difloxacin, flumequine and marbofloxacin) were determined in milk and egg samples by a simplified high-performance liquid chromatographic procedure using a micellar mobile phase. No extraction was needed to precipitate the proteins from the matrices since they were solubilised in micelles. The only pretreatment steps required were homogenisation, dilution and filtration before injecting the sample into the chromatographic system. An adequate resolution of the quinolones was achieved by a chemometrics approach where retention was modelled as a first step using the retention factors in only five mobile phases. Afterwards, an optimisation criterion was applied to consider the position and shape of the chromatographic peaks. Analytical separation involved a C18 reversed-phase column, a hybrid micellar mobile phase of 0.05 M sodium dodecyl sulphate, 10% (v/v) butanol and 0.5% (v/v) triethylamine buffered at pH 3 and fluorimetric detection. Quinolones were eluted in less than 15 min without the protein band or other endogenous compounds from the food matrices interfering. The calculated relevant validation parameters, e.g., decision limit (CC(α)), detection capability (CC(ß)), repeatability, within-laboratory reproducibility, recoveries and robustness, were acceptable and complied with European Commission Decision 2002/657/EC. Finally, the proposed method was successfully employed in quantifying the four quinolones in spiked egg and milk samples.

Effect of short-chain alcohols on surfactant-mediated reversed-phase liquid chromatographic systems.

The behaviour of ß-blockers in a reversed-phase liquid chromatographic (RPLC) column with mobile phases containing a short-chain alcohol (methanol, ethanol or 1-propanol), with and without the surfactant sodium dodecyl sulphate (SDS), was explored. Two surfactant-mediated RPLC modes were studied, where the mobile phases contained either micelles or only surfactant monomers at high concentration. Acetonitrile was also considered for comparison purposes. A correlation was found between the effects of the organic solvent on micelle formation (monitored by the drop weight procedure) and on the nature of the chromatographic system (as revealed by the retention, elution strength and peak shape of ß-blockers). When SDS is added to the mobile phase, the free surfactant monomers bind the C18 bonded chains on the stationary phase, forming an anionic layer, which attracts strongly the cationic ß-blockers. The retention is modified as a consequence of the solving power of the organic solvent, micelles and surfactant monomers. The molecules of organic solvent bind the micelles, modify their shape, and may avoid their formation. They also bind the monomers of surfactant, desorbing them from the stationary phase, which affects the retention. The remaining surfactant covers the free silanols on the siliceous support, avoiding the interaction with the cationic solutes. The retention of ß-blockers results from a combination of electrostatic and hydrophobic interactions, the latter being weaker compared to the hydro-organic system. The peak efficiencies and asymmetries are excellent tools to probe the surfactant layer on the stationary phase in an SDS/organic solvent system. The peaks will be nearly symmetrical wherever enough surfactant coats the stationary phase (up to 60% methanol, 40% ethanol, 35% 1-propanol, and 50% acetonitrile).

Performance of short-chain alcohols versus acetonitrile in the surfactant-mediated reversed-phase liquid chromatographic separation of ß-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 ß-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 ß-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 ß-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 ß-blockers, owing to the poorer selectivity and wider peaks.

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 (beta-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.

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

The behaviour of a reversed-phase liquid chromatographic (RPLC) system (i.e. elution order, resolution and analysis time), used in the analysis of beta-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 x 10(-3)-0.15M 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 x 10(-3)M), and more than 30% acetonitrile. High submicellar RPLC appeared as the most promising mode, as it allowed full resolution of the beta-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.

Countercurrent chromatography: people and applications.

The scientific literature was scanned for the published research articles dealing with countercurrent chromatography (CCC) over the time period 1980-May 2008. The search returned 1638 articles that were analyzed focussing on people and applications. Concerning the people, it was found that the geographical location of the CCC authors was relatively well balanced between USA, Asia with mainly China and Japan and Europe. Yoichiro Ito, the inventor of the technique, is by far the most productive author in the field with 331 articles or more than one over five CCC articles published in the time period. Without surprise, English is the dominant language with more than 82% of the articles. A significant 8% amount of CCC articles were published in Chinese in Chinese journals. Chromatography journals are the logical tribune for half of the published CCC articles. Concerning the applications, the separation and purification of natural compounds is the dominant theme in CCC making the subject of more than one article over two. Starting from the plant extract, CCC in few steps can produce significant amounts of more than 95% pure compounds used for identification and/or property studies. Other applications are found in the pharmaceutical and chemical field. The separation of enantiomers on the preparative scale is a field of growing importance.

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.