Conventional Reversed-phase Liquid Chromatography Research Articles

Chromatographic behavior of polar compounds in the transition between reversed-phase and submicellar/micellar liquid chromatography

Micellar liquid chromatography (MLC) is a reversed-phase liquid chromatography (RPLC) mode employing a surfactant, typically sodium dodecyl sulfate (SDS), which is biodegradable, in an aqueous or aqueous-organic mobile phase above its critical micellar concentration (CMC). Under these conditions, substantial alterations in retention, selectivity and peak profile occur in comparison to RPLC using conventional hydro-organic mobile phases. These changes arise from the presence of micelles in the mobile phase and the adsorption of surfactant on the stationary phase. In this work, the chromatographic performance of polar compounds of diverse nature is investigated using mobile phases containing SDS below and above its CMC in the presence of acetonitrile. Three nucleosides, four diuretics, and four sulfonamides, all of biomedical and pharmaceutical significance and possessing distinct acid-base characteristics, were chosen as probe compounds. Modifications in retention and peak shape were investigated using experimental conditions that enable the transition from submicellar to micellar conditions. A comparison was also made with conventional RPLC. Analysing the polar compounds with RPLC under low submicellar or micellar conditions significantly decreases the consumption of organic solvent in comparison to hydrophilic liquid chromatography (HILIC).

Aqueous liquid chromatography with mobile phases of sodium dodecyl sulphate and ionic liquid

In conventional reversed-phase liquid chromatography (RPLC) with hydro-organic solvents, basic cationic solutes yield retained, broad, asymmetric peaks, owing to their interaction with free anionic silanols in the stationary phase. RPLC mobile phases to which the anionic surfactant sodium dodecyl sulphate (SDS), or an ionic liquid (IL) are added, have been proposed as solutions, since these additives are able to block the silanol effect thus improving the chromatographic performance. With these additives, it is however necessary to increase the elution strength by adding an organic solvent, such as an alcohol or acetonitrile. A novel aqueous liquid chromatographic mode (in the absence of organic solvent) is here proposed, where the mobile phases contain only a mixture of aqueous solutions of SDS and an IL derived from 1-alkyl-3-methylimidazolium associated to chloride, both environmentally friendly. When these reagents are added, the anionic surfactant adsorbed on the stationary phase is able to attract the cationic solutes, whereas the adsorbed IL cation repels them. The combination of both effects (attraction and repulsion) allows the modulation of retention, by varying the IL/SDS ratio. Given the character of the additives, a type of green liquid chromatography is achieved. In this work, the chromatographic behavior of six basic compounds of pharmaceutical interest, the β-adrenoceptor antagonists acebutolol, atenolol, carteolol, metroprolol, oxprenolol and propranolol, is examined. In order to assess the chromatographic behavior of the mixed mobile phases containing SDS and IL, changes in retention, peak profile and resolution of mixtures of the analytes were explored at varying concentration of the additives.

A Combination of Native LC-MS Approaches for the Comprehensive Characterization of the Antibody-Drug Conjugate Trastuzumab Deruxtecan.

Native mass spectrometry (nMS) approaches appear attractive to complement bottom-up strategies traditionally used in biopharmaceutical industries thanks to their quite straightforward and rapid workflows, especially through online hyphenation of non-denaturing liquid chromatography (LC) to nMS. The present work provides an overview of the state-of-the-art chromatographic tools available for the detailed characterization of monoclonal antibody (mAb) formats, exemplified on the antibody-drug conjugate (ADC) trastuzumab deruxtecan (T-DXd). T-DXd was first characterized by conventional reversed phase LC (rpLC) and peptide mapping. Couplings of size exclusion chromatography (SEC), cation exchange chromatography (CEX), and hydrophobic interaction chromatography (HIC) to nMS were used to gain further insights into size, hydrophobic, and charge variants of T-DXd and its parental mAb trastuzumab, at intact and middle-up levels. SEC-nMS first offered a direct snapshot of the homogeneous conjugation of T-DXd, with an average drug-to-antibody ratio (DAR) of 8 in agreement with a conjugation on cysteines after reduction of all interchain disulfide bonds. Moreover, SEC-nMS afforded precise identification and quantification of aggregates and fragments. Middle-up level experiments performed after IdeS digestion confirmed that drug conjugation occurs in the Fab region of the mAb, as seen with rpLC. HIC separated two DAR8 species that could not be differentiated by nMS. Although middle-up HIC-nMS proved to be more informative for oxidized forms, the identification of minor variants was still difficult because of poor MS signal quality, showing how the coupling of HIC to nMS remains challenging. Lastly, middle-up CEX-nMS provided accurate determination and localization of post-translational modifications, with several acidic/basic variants within Fab and Fc regions of T-DXd that were also identified by peptide mapping. This study illustrates the strengths and drawbacks of each LC-nMS coupling. By combining SEC-, HIC-, and CEX-nMS, we were able to achieve a comprehensive characterization of T-DXd without extensive sample preparation prior to MS analysis.

Solvent strength of aqueous phase for two typical biphasic solvent systems in high-speed countercurrent chromatography

In the present work, influence of solvent strength of aqueous phase for two frequently-used biphasic solvent system in partition coefficient (K) of selected solutes were mainly studied, and a new method for selection of biphasic solvent system was proposed for high-speed countercurrent chromatographic separations. Solvent strength was referred to the typical theory that was deeply investigated in conventional reversed-phase liquid chromatography. Experimental results showed that a linear relationship between log(K) of solutes and apparent content of methanol in biphasic solvent system was found for the biphasic solvent system hexane-ethyl acetate-methanol-water (HEMWat), which was consistent with the relationship between real content and apparent content of methanol in this system. Meanwhile, a quadratic relationship was found between log(K) of solutes and apparent content of methanol in biphasic solvent system chloroform-methanol-water (ChMWat), in which it was found that the relationship between real content and apparent content of methanol in this system was also quadratic. In addition, a visual and simple method was proposed to select a suitable biphasic solvent system for separation of target compounds by high-speed countercurrent chromatography with isocratic elution, which saves a lot of manpower and material resources in order to find a suitable two-solvent system. An optimal biphasic solvent system for isolation of several tested compounds by high-speed countercurrent chromatography was easily obtained using our proposed method.

Separation of chiral and achiral impurities in paroxetine hydrochloride in a single run using supercritical fluid chromatography with a polysaccharide stationary phase

Separating paroxetine hydrochloride and its impurities using conventional reversed-phase liquid chromatography (RPLC) is challenging due to their highly similar structures. In the present study, a rapid, simple, sensitive and environmentally friendly method was developed for the determination of chiral and achiral impurities in raw materials of paroxetine hydrochloride using chiral supercritical fluid chromatography (SFC). The impacts of chiral stationary phases (CSPs), mobile phases, column temperature and back pressure on the retention and separation of analytes were comprehensively evaluated. After method optimization, a satisfying result was obtained on a cellulose tris-(3-chloro-4-methylphenylcarbamate) stationary phase in 4.0 min using 70% CO2 and 20 mM ammonium acetate in 30% methanol as the mobile phase. Molecular docking was further performed to understand the interactions between the analytes and CSP. The results suggested that hydrogen bonding and π–π interactions were the dominant interactions. The affinity given by the software was in good agreement with the elution order and free energy (△G) values obtained from van’t Hoff equations. The results of molecular docking also provide insights into the different retentions of N-methylparoxetine at different temperatures. The results of method validation revealed that the method was sensitive with a limit of detection of approximately 0.05 μg·mL−1 (corresponding to approximately 0.005% paroxetine hydrochloride in the sample solution). The relative standard deviations (RSDs) of precision and intra-assay precision were all less than 2.0%, and the recoveries of the method were 93.8~105.3% with RSDs less than 3.0%. The chiral and achiral RPLC methods included in the Chinese pharmacopoeia and the SFC method proposed in this study were simultaneously used to determine the impurity content in the raw materials of paroxetine hydrochloride. The results showed that impurities that cannot be detected by the reference method can be accurately quantified using the SFC method. In addition, the SFC method has advantages in terms of throughput, analysis cost and simplicity. This study can provide a reference for further research of impurities in paroxetine hydrochloride and promote the application of chiral SFC in the rapid separation of structurally similar compounds.

Reducing metal-ion mediated adsorption of acidic peptides in RPLC-based assays using hybrid silica chromatographic surfaces

In this study we evaluate column hardware exhibiting a novel hybrid silica surface in its ability to mitigate metal-induced adsorption artifacts such as chromatographic peak tailing for acidic amino acid residue containing peptides. Using a conventional reversed-phase liquid chromatography (RPLC)-based method, chromatographic performance of a peptide map was compared using a traditional stainless-steel column and an equivalent column bearing a novel hybrid silica surface. Tailing factors for six peptides containing acidic amino acid residues (Tf ≥ 1.50) were observed to be reduced up to 80% to a nominal value Tf ≤ 1.2 with R.S.D. % ≤ 4%. Furthermore, recovery for two of the identified peptides exhibited increased recovery in addition to reduced peak tailing when using the column bearing the hybrid silica surface. Performance was unaffected for peaks where there were no implications of metal induced effects. Collectively, this study demonstrates that the novel hybrid silica surface can effectively reduce peak tailing for acidic residue containing peptides.

Paper spray mass spectrometry - A potential complementary technique for the detection of polar compounds in sports drug testing.

In this proof-of-concept study, paper spray mass spectrometry was investigated as a high-throughput and fully automated technique for the initial testing of particularly polar compounds that are prohibited in sports. The technique allows the ionization of analytes from complex sample matrices such as blood and urine when spotted onto a paper strip. By minimizing sample preparation and omitting chromatographic separation, paper spray mass spectrometry benefits from considerable cost- and time-savings compared with conventional high performance liquid chromatography/tandem mass spectrometry, especially in cases where conventional reversed-phase liquid chromatography offers limited applicability. All but one of the investigated model compounds fulfilled the World Anti-Doping Agency's (WADA's) requirements regarding the applicable minimum required performance limits for initial testing procedures. In addition, the combination of paper spray mass spectrometry and ion mobility separation results in enhanced selectivity and sensitivity and is a particularly valuable analytical configuration.

Simultaneous analysis of multiple oligonucleotides by temperature-responsive chromatography using a poly(N-isopropylacrylamide)-based stationary phase

Oligonucleotide therapeutics have contributed remarkably to healthcare, being well suited for the treatment of intractable diseases that are difficult to approach using conventional drug modalities. However, as common techniques of oligonucleotide analysis rely on reversed-phase or ion-exchange liquid chromatography and thus employ toxic organic solvents and/or ion-pairing reagents, better alternatives are highly sought after. Poly(N-isopropylacrylamide) (PNIPAAm) is widely used in temperature-responsive chromatography (TRC), which relies on column temperature variation to control the physical properties of the stationary phase and, unlike conventional reversed-phase liquid chromatography, avoids the use of toxic organic solvents and complicated gradient methods. Herein, PNIPAAm copolymer hydrogel-modified silica beads were used for the simultaneous analysis of multiple synthetic oligonucleotides by TRC to recognize differences in the length of single nucleotides, single bases, and the number of phosphorothioated sites. Temperature-responsive elution was observed in all cases. Each separation of all combinations of multiple oligonucleotides was better at higher temperatures above the lower critical solution temperature and was performed without the use of organic solvents and gradient methods. In the case of multiply phosphorothioated oligonucleotides, good separation was achieved using an aqueous solvent and isocratic elution in the absence of ion-pairing reagents. Thus, the developed procedure was concluded to be well suited for oligonucleotide analysis.Graphical abstract

Isocratic micellar liquid chromatography using mixed anionic and non-ionic surfactants as mobile phase additives for separation of 17 free amino acids

In this work, an isocratic micellar chromatographic method for separation of 17 free amino acids was first established. Mixed anionic and non-ionic surfactants were used as mobile phase additives. Besides, a chromatographic environment was simulated and the possible mechanism was discussed. Pre-column derivatization with 9-fluorenylmethyl chloroformate was used before chromatographic analysis. The optimized chromatographic conditions were the mobile phase A (0.075 M sodium dodecyl sulfate solutions and 0.010 M polyoxyethylene lauryl ether containing 20 mM ammonium acetate at pH 3.5) and B (100% acetonitrile) (85:15, v/v) running at 1.2 mL/min by a Venusil XBP C18 column (5 µm, 250 × 4.6 mm) at 35.0 °C. Compared with the conventional reversed phase liquid chromatography, the consumption of organic solvent in the method is lower which reduces the analysis cost and being environment-friendly. In addition, complex gradient elution is not required. This provides an alternative way for the separation of amino acids.

A method to directly analyze free-drug–related species in antibody-drug conjugates without sample preparation

Residual free-drug–related species that are present in antibody-drug conjugates (ADC) are a potential safety risk to patients and are therefore categorized as a critical quality attribute that must be strictly monitored and controlled. Among the many analytical methods developed for free-drug analysis, reversed-phase liquid chromatography (RP-LC) is the most common approach. Conventional RP-LC methods for free-drug analysis, however, involve labor-intensive sample preparation. Here we present a new RP-LC method to directly analyze free-drug–related species in an ADC sample without the need for sample preparation. In our work, free-drug–related species were very well separated from ADC peaks in the chromatography gradient. Typical performance issues observed in conventional RP-LC, such as column fouling, detection interference, and carryover, were not observed or were negligible with this new method. Three options were evaluated for free-drug quantitation: Strohl (2017) [1] use of an external free drug calibration curve for determination of absolute concentration; Perez et al. (2014) [2] calculation of relative percentage based on peak area ratio between free drug and ADC at a characteristic wavelength unique for drug payload; and (Beck et al., 2017) [3] calculation of relative percentage based on peak area ratio between free drug and corrected ADC peak area (at any wavelength). The method with calibration curve provides the highest sensitivity, the best accuracy and precision for determination of free drug present in the ADC. However, the second and third options were simpler because they eliminated the need for an external calibration curve, making them worth exploring. Due to its simplicity and compatibility with mass spectrometry, the new method is also a good choice for direct analysis of stability samples.

(S)-1-(1-Methylpyridin-2-yl)-3-aminopiperidine as a novel derivatization reagent capable of enantiomeric separation and enhanced ESI-MS/MS detection for chiral carboxylic acids

A novel derivatization reagent, (S)-1-(1-methylpyridin-2-yl)-3-aminopiperidine (MPAPp), was designed and developed for the enantiomeric separation by conventional reversed-phase liquid chromatography (LC) and the sensitive electrospray ionization-tandem mass spectrometry (ESI-MS/MS) detection of chiral carboxylic acids. MPAPp reacted with the carboxylic acids at 60 °C within 5 min in the presence of a condensation agent to produce the positively-charged and diastereomeric derivatives. The MPAPp-derivatization enabled the satisfactory enantiomeric separation not only of the α- and β-chiral carboxylic acids [ketoprofen (KET), etodolac and 3-hydroxypalmitic acid], but also of the γ-chiral carboxylic acid [2-(γ-carboxyethyl)-6-hydroxy-2,7,8-trimethylchroman (γ-CEHC)] with the resolution (Rs) values of 1.35–1.82. The derivatization also worked for the enantiomeric separation of the ε-chiral carboxylic acid [α-lipoic acid, separation factor (α) = 1.04]. The detection responses of the MPAPp-derivatives were 25–500-times greater than the intact carboxylic acids, therefore, the low femtomolar derivatives were fully detected. The trace detection of a γ-CEHC enantiomer (limit of detection, 1.9 fmol on column) in saliva was achieved by the MPAPp-derivatization followed by LC/ESI-MS/MS. The pharmacokinetic profiles of the KET enantiomers during/after the MOHRUS® patch application were also obtained from the saliva analysis using the MPAPp-derivatization based LC/ESI-MS/MS method, which was precise (intra- and inter-assay relative standard deviations, <6.6% and <4.6%, respectively) and accurate (96.2–105.3%) within the range of 35–280 pg/mL for each enantiomer.

Search of non-ionic surfactants suitable for micellar liquid chromatography.

Most reports in reversed-phase liquid chromatography (RPLC) with micellar mobile phases make use of the anionic sodium dodecyl sulfate. This surfactant masks efficiently the silanol groups that are the origin of the poor efficiencies and tailing peaks observed for basic compounds in conventional RPLC. However, it has the handicap of yielding excessive retention, which forces the addition of an organic solvent to reduce the retention times to practical values. Other surfactants, such as the non-ionic polyoxyethylene(23)lauryl ether (Brij-35), are rarely used. Brij-35 allows the separation of a large range of analytes in adequate retention times, without the need of adding an organic solvent to the mobile phase. However, this non-ionic surfactant shows irreversible adsorption on chromatographic columns and peak shape is poorer. Therefore, the search of non-ionic surfactants with similar properties to Brij-35, but showing reversible adsorption and better peak shape, can be of great interest. In this work, the adequacy of several non-ionic surfactants as modifiers in RPLC has been explored, being polyoxyethylene(10)tridecyl ether particularly attractive. The separation of different types of compounds was checked: sulfonamides (acidic), β-adrenoceptor antagonists and tricyclic antidepressants (basic with diverse polarity), and flavonoids (with and without hydroxyl groups on the aromatic rings). The chromatographic behaviors were examined in terms of retention and peak shape. The results were compared with those obtained with Brij-35.

Comparative study of two different chromatographic approaches for quantitation of hydrocortisone acetate and pramoxine hydrochloride in presence of their impurities

In the present study, we compare the performance of two reversed-phase liquid chromatographic approaches using different eluents either conventional hydro-organic eluent or micellar one for simultaneous estimation of hydrocortisone acetate and pramoxine hydrochloride in presence of their degradants and process-related impurities; hydrocortisone and 4-butoxyphenol, respectively. For conventional reversed-phase liquid chromatography (RPLC), separation of the studied compounds was completed on an Inertsil ODS 3-C18 column (150 mm × 4.6 mm, 5 μm particle size) with a mobile phase consists of 50 mM phosphate buffer (pH 5.0): acetonitrile (50: 50, v/v). For micellar liquid chromatography (MLC), an Eclipse XDB-C8 column (150 mm × 4.6 mm, 5 μm particle size) was chosen for the separation with a green mobile phase consists of 0.15 M sodium dodecyl sulfate, 0.3% triethylamine and 10% n-butanol in 20 mM orthophosphoric acid (pH 5.0). Both methods were extended to analyze hydrocortisone acetate and pramoxine hydrochloride in their co-formulated cream. RPLC was superior to MLC with regard to sensitivity for the estimation of impurities. While, MLC represents an eco-friendly, less hazardous and biodegradable approach. Furthermore, the direct injection of the cream to the system without the need to laborious samples pretreatment, excessive amount of analysis time and/or use of large amount of toxic organic solvents is one of the outstanding advantages of MLC.

Separation and characterization of chemical constituents in Ginkgo biloba extract by off-line hydrophilic interaction × reversed-phase two-dimensional liquid chromatography coupled with quadrupole-time of flight mass spectrometry

Ginkgo biloba extract (GBE), derived from the leaves of Ginkgo biloba L., is one of the most widely used traditional Chinese medicines worldwide. Due to high structural diversity and low abundance of chemical constituents in GBE, conventional reversed-phase liquid chromatography has limited power to meet the needs of its quality control. In this study, an off-line hydrophilic interaction×reversed-phase two-dimensional liquid chromatography (HILIC×RP 2D-LC) system coupled with diode array detection (DAD) and quadrupole time-of-flight mass spectrometry (qTOF-MS) was established to comprehensively analyze the chemical constituents of GBE. After optimizing the chromatographic columns and mobile phase of 2D-LC, a Waters XBridge Amide column using acetonitrile/water/formic acid as the mobile phase was selected as the first dimension to fractionate GBE, and the obtained fractions were further separated on an Agilent Zorbax XDB-C18 column with methanol/water/formic acid as the mobile phase. As a result, a total of 125 compounds were detected in GBE. The orthogonality of the 2D-LC system was 69.5%, and the practical peak capacity was 3864 and 2994, respectively, calculated by two different methods. The structures of 104 compounds were tentatively characterized by qTOF-MS analysis, and 21 of them were further confirmed by comparing with reference standards. This established HILIC×RP 2D-LC-qTOF/MS system can greatly improve the separation and characterization of natural products in GBE or other complicated herbal extracts.

Analysis of catecholamines in urine by unique LC/MS suitable ion-pairing chromatography.

The catecholamines, epinephrine (E) and norepinephrine (NE) are small polar, hydrophilic molecules, posing significant challenges to liquid chromatography - tandem mass spectrometry (LC-MS/MS) method development. Specifically, these compounds show little retention on conventional reversed-phase liquid chromatography columns. This work presents development and validation of an LC-MS/MS method for determining catecholamines in urine, based on a new approach to ion-pairing chromatography (IPC), in which the ion-pairing reagent (IPR), 1-Heptane Sulfonic Acid (HSA), is added to the extracted samples instead of the mobile phases. A Hamilton STARlet workstation carried out the solid phase extraction of urine samples. The extracted samples were diluted with 60mmol/L HSA and injected on a Kinetex core-shell biphenyl column with conventional LC-MS/MS suitable mobile phases. Chromatographic separation of E and NE was achieved successfully with very stable retention times (RT). In 484 injections, the RTs were steady with a CV of less than ±4%. Furthermore, HSA was separated from E and NE, allowing HSA to be diverted to waste instead of entering the mass spectrometer ion chamber. The method was validated with good analytical performance, and even though the analysis for urinary catecholamines is increasingly being replaced by plasma free metanephrines in diagnosing pheochromocytomas, this work represents the application of a new analytical technique that can be transferred to other small polar molecules, that are difficult to chromatograph on traditional reversed phase columns.

Development of a sensitive and rapid method for rifampicin impurity analysis using supercritical fluid chromatography

A novel simple, fast and efficient supercritical fluid chromatography (SFC) method was developed and compared with RPLC method for the separation and determination of impurities in rifampicin. The separation was performed using a packed diol column and a mobile phase B (modifier) consisting of methanol with 0.1% ammonium formate (w/v) and 2% water (v/v). Overall satisfactory resolutions and peak shapes for rifampicin quinone (RQ), rifampicin (RF), rifamycin SV (RSV), rifampicin N-oxide (RNO) and 3-formylrifamycinSV (3-FR) were obtained by optimization of the chromatography system. With gradient elution of mobile phase, all of the impurities and the active were separated within 4min. Taking full advantage of features of SFC (such as particular selectivity, non-sloping baseline in gradient elution, and without injection solvent effects), the method was successfully used for determination of impurities in rifampicin, with more impurity peaks detected, better resolution achieved and much less analysis time needed compared with conventional reversed-phase liquid chromatography (RPLC) methods.

Screening and confirmation of myo-inositol trispyrophosphate (ITPP) in human urine by hydrophilic interaction liquid chromatography high resolution / high accuracy mass spectrometry for doping control purposes.

Myo-inositol trispyrophosphate (ITPP) is a novel allosteric effector of haemoglobin with high permeation selectivity across the red blood cell plasma membrane. Due to its potential to reduce the oxygen affinity of haemoglobin, ITPP application results in an enhanced oxygen release in hypoxic tissues. Therefore, ITPP is being examined for the treatment of numerous illnesses that involve hypoxia, such as cardiovascular diseases, cancer or Alzheimer's disease. Similar to the prohibited substance Efaproxiral®, ITPP increases maximal exercise capacity in mice, providing high potential to be misused in sports. To keep up with cheating athletes, a fast and reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for screening and confirmation of ITPP in human urine for doping control purposes was developed. According to the molecule's distinct hydrophilic properties, extraction from complex biological matrices is challenging and conventional reversed phase liquid chromatography (RPLC) separations are not suitable for its detection. Therefore an approach based on hydrophilic interaction liquid chromatography (HILIC) Orbitrap mass spectrometry was established. The methodology was fully validated for qualitative purposes. Screening and confirmation assay are characterized by satisfactory specificity and robustness, adequate intra-day (screening: 4.9-8.1%; confirmation: 2.0-6.7%) and inter-day precision (screening: 4.6-9.1%; confirmation: 1.8-6.6%), excellent linear correlations (>0.99) with sufficient LLOD in the sub ng/mL range (screening: 15 ng/mL; confirmation: 1 ng/mL). In addition it could be shown that ITPP is stable in human urine under the mandatory storage period and conditions for doping control laboratories. To our knowledge, this is the first validated 'dilute-and-inject' LC-MS/MS method for the reliable detection of ITPP in human urine.

Implementation of gradients of organic solvent in micellar liquid chromatography using DryLab®: Separation of basic compounds in urine samples

In micellar liquid chromatography (MLC), chromatographic peaks are more evenly distributed compared to conventional reversed-phase liquid chromatography (RPLC). This is the reason that most procedures are implemented using isocratic elution. However, gradient elution may be still useful in MLC to analyse mixtures of compounds within a wide range of polarities, decreasing the analysis time. Also, it benefits the determination of moderately to low polar compounds in physiological fluids performing direct injection: an initial micellar eluent with a low organic solvent content, or a pure micellar (without surfactant) solution, will provide better protection of the column against the proteins in the physiological fluid, and once the proteins are swept away, the elution strength can be increased using a positive linear gradient of organic solvent to reduce the analysis time. This work aims to encourage analysts to implement gradients of organic solvent in MLC, which is rather simple and allows rapid analytical procedures without pre-treatment or the need of re-equilibration. The implementation of gradient elution is illustrated through the separation of eight basic compounds (β-blockers) in urine samples directly injected into the chromatograph, the most hydrophobic showing large retention in both conventional RPLC and MLC. The use of the DryLab® software to optimise gradients of organic solvent with eluents containing a fixed amount of surfactant above the critical micellar concentration is shown to provide satisfactory predictions, and can facilitate greatly the implementation of gradient protocols.

High Submicellar Liquid Chromatography

Surfactant addition above the critical micellar concentration (CMC), in reversed-phase liquid chromatography (RPLC), was proposed as a way to modify the selectivity and analysis time, giving rise to a chromatographic mode called micellar liquid chromatography (MLC). However, solutions containing only surfactant are too weak and yield poor peak shape. This was remediated by the addition of a small amount of organic solvent. To preserve the existence of micelles, in MLC high contents of organic solvent are avoided. Nevertheless, there is no reason to neglect the potentiality of mobile phases containing a surfactant above its CMC in water and a high organic solvent content (without micelles). This chromatographic mode has been called high submicellar liquid chromatography (HSLC). Several reported procedures show that the combination of stronger elution strength, larger selectivity and improved peak shape, with respect to MLC and conventional RPLC, makes HSLC a promising chromatographic mode to achieve in practical times separations of compounds unresolved or highly retained with other RPLC modes. Some insights on the interactions that occur inside the chromatographic column, the modification of the stationary and mobile phases, retention modeling, and chromatographic performance in HSLC are here offered, in comparison to MLC and conventional RPLC.

Basic Principles of MLC

Micellar liquid chromatography (MLC) is an efficient alternative to conventional reversed-phase liquid chromatography with hydro-organic mobile phases. Almost three decades of experience have resulted in an increasing production of analytical applications. Current concern about the environment also reveals MLC as an interesting technique for “green” chemistry because it uses mobile phases containing 90% or more water. These micellar mobile phases have a low toxicity and are not producing hazardous wastes. 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 a great variety of interactions (hydrophobic, ionic, and steric) with major implications in retention and selectivity. From its beginnings in 1980, the technique has evolved up to becoming in a real alternative in some instances (and a complement in others) to classical RPLC with aqueous-organic mixtures, owing to its peculiar features and unique advantages. The addition of an organic solvent to the mobile phase was, however, soon suggested in order to enhance the low efficiencies and weak elution strength associated with the mobile phases that contained only micelles.