Selectivity In Reversed-phase Liquid Chromatography Research Articles

Deep eutectic solvents-derivated carbon dots-decorated silica stationary phase with enhanced separation selectivity in reversed-phase liquid chromatography

In this work, deep eutectic solvents-based carbon dots (DESCDs) were prepared and bonded to the silica surface for the first time to form a new hydrophobic chromatographic stationary phase (Sil-DESCDs). The successful preparation of DESCDs and Sil-DESCDs were demonstrated by a series of characterizations including transmission electron microscopies, laser scanning confocal microscope, Fourier transform infrared spectrometry, elemental analysis, etc. Retention behavior of Sil-DESCDs was evaluated using Tanaka and Engelhardt standard test mixtures. The results showed that this new stationary phase had excellent separation performance for polycyclic aromatic hydrocarbons, flavonoids, aromatic amines and phenolic compounds. Excellent separation selectivity for the 3-phenylene ring isomers including phenanthrene and anthracene, the 4-phenylene ring isomers including pyrene, triphenylene, chrysene and 1,2-benzanthracene was also obtained. Especially, prednisolone and hydrocortisone, which have very similar structures, can be separated using pure water as the mobile phase. In addition, the flavonoids in Astragalus extracts including calycosin-7-glucoside, ononin, calycosin and formononetin were determined using this new column, their concentrations were 0.050, 0.031, 0.023 and 0.034 mg/mL, respectively.

Selection of calibration compounds for selectivity evaluation of siloxane-bonded silica columns for reversed-phase liquid chromatography by the solvation parameter model

For the faster evaluation of selectivity in reversed-phase liquid chromatography of siloxane-bonded silica columns using the solvation parameter model a minimal set of calibration compounds is described suitable for mobile phase composition from 20-70% (v/v) methanol-, acetonitrile-, or tetrahydrofuran-water. The Kennard-Stone uniform mapping algorithm is used to select the calibration compounds from a larger database of compounds with known retention properties used earlier for column selectivity evaluation. Thirty-five compounds are shown to be necessary to minimize the standard deviation of the system constants and to minimize the difference between the system constants determined by conventional calibration and the values obtained for the reduced calibration compounds. The models for SunFire C18 with methanol-, acetonitrile- and tetrahydrofuran-water mobile phase compositions and XBridge Shield RP18, XBridge C8, XBridge Phenyl and Discovery HS F5 with methanol- and acetonitrile-water mobile phase compositions had an average coefficient of determination of 0.996 (standard deviation = 0.003, n = 11) and average standard error of the estimate 0.025 (standard deviation = 0.005, n = 11) for the reduced calibration compounds. Some octadecylsiloxane-bonded silica stationary phases with a high bonding density and methanol-water mobile phase compositions containing ≤ 30% (v/v) methanol exhibit extreme retention factors (log k > 2.5) for the low-polarity, two-ring aromatic compounds in the thirty-five compound calibration set. Alternative calibration compounds with more favorable retention properties are suggested as replacements in these cases. The predictive capability of the calibration models is validated using external test sets characterized by the average error, average absolute error and root mean square error of prediction. For the thirty-five calibration compounds sets the average absolute error 0.026 (standard deviation = 0.009, n = 11) and root mean square error of prediction 0.032 (standard deviation = 0.010, n = 11) confirm the suitability of the calibration models for column selectivity evaluation. System maps for XBridge Shield RP18 for 20-70% (v/v) methanol-water and Synergi Hydro-RP and 50% (v/v) methanol-water at temperatures from 25-65 °C together with a correlation diagram for XBridge Shield RP18 and SunFire C18 are presented as representative applications of the reduced calibration compounds for column selectivity evaluation.

Intrinsic difference between phenyl hexyl- and octadecyl-bonded silicas in the solute retention selectivity in reversed-phase liquid chromatography with aqueous mobile phase.

For choosing an optimal column for a particular separation by reversed-phase liquid chromatography (RPLC), it is essential to quantitatively understand the effects of the chemical structure of hydrophobic bonded layer derived onto silica particles on the distribution equilibrium of a solute compound at the interface between the aqueous mobile phase and the packing material. However, there is still a lack of understanding of the solute distribution equilibrium in RPLC separation due to the complexities of the chemistry at the interface between the mobile phase and the bonded layer. We successfully determined the distribution coefficients of various organic compounds concerning to their accumulation onto the water/bonded layer interface and into the bonded layer from bulk water using surface-bubble-modulated liquid chromatography with octadecyl- and phenyl hexyl-bonded silica columns. The water/phenyl hexyl-bonded layer interface accumulates organic compounds much less than the water/octadecyl-bonded layer interface due to its lower interfacial tension, and this result suggests that phenyl hexyl group orient their benzene ring facing toward water. On the other hand, aromatic moiety of phenyl hexyl group enhances partitioning of the organic compounds into the bonded layer. Experimental findings in the present work demonstrated that the water/bonded layer interface and the bonded layer itself have independent contributions to the solute distribution and the water/phenyl hexyl-bonded layer interface shows quite different solute retention selectivity from the water/octadecyl-bonded layer interface.

Tuning retention and selectivity in reversed-phase liquid chromatography by using functionalized multi-walled carbon nanotubes

Aim of this work was to explore the possibility of retention and selectivity tuning in reversed-phase liquid chromatography by means of chemically modified multi-walled carbon nanotubes (MWCNTs). These were synthesized by derivatizing pristine MWCNTs with amino-terminated alkyl chains containing polar embedded groups. A novel hybrid material based on functionalized MWCNTs (MWCNTs-R-NH2) was prepared, characterized and tested. The idea was to design a mixed-mode separation medium basing its sorption properties on the peculiar characteristics of MWCNTs combined with the chemical interactions provided by the functional chains introduced on the nanotube skeleton. MWCNTs-R-NH2 were easily grafted to silica microspheres by gamma radiation (using a 60Co source) in the presence of polybutadiene as the linking agent. The composite was characterized by scanning electron microscopy (SEM) and Brunauer, Emmett and Teller (BET) analysis in terms of structural morphology, surface area and porosity. The MWCNTs-R-NH2 sorbent was tested as stationary phase. The reversed-phase behaviour was first proved by analysis of alkylbenzenes, while the key role of CNT derivatization in addressing the selectivity/affinity towards the solutes was evidenced by testing three classes of analytes, viz. barbiturates, steroid hormones and alkaloids. These compounds, with different molecular structure and polarity, were here analysed for the first time on CNT-based LC stationary phases. The behaviour of the novel sorbent was compared in terms of retention capability and resolution with that observed using unmodified MWCNTs, pointing out the mixed-mode characteristics of the MWCNTs-R-NH2 material. The same test mixtures were analysed also on a conventional mono-modal separation sorbent (C18) to highlight the particular behaviour of the (derivatized)MWCNTs-based stationary phases. The novel material showed better performance in separation of polar compounds, i.e. barbiturates and alkaloids, than the unmodified MWCNTs and than the C18 column. Results showed that MWCNT functionalization is powerful to modulate retention/selectivity in reversed-phase liquid chromatography.

Retention mechanism for polycyclic aromatic hydrocarbons in reversed-phase liquid chromatography with monomeric stationary phases

Reversed-phase liquid chromatography (RPLC) is the foremost technique for the separation of analytes that have very similar chemical functionalities, but differ only in their molecular shape. This ability is crucial in the analysis of various mixtures with environmental and biological importance including polycyclic aromatic hydrocarbons (PAHs) and steroids. A large amount of effort has been devoted to studying this phenomenon experimentally, but a detailed molecular-level description remains lacking. To provide some insight on the mechanism of shape selectivity in RPLC, particle-based simulations were carried out for stationary phases and chromatographic parameters that closely mimic those in an experimental study by Sentell and Dorsey [J. Chromatogr. 461 (1989) 193]. The retention of aromatic hydrocarbons ranging in size from benzene to the isomeric PAHs of the formula C(18)H(12) was examined for model RPLC systems consisting of monomeric dimethyl octadecylsilane (ODS) stationary phases with surface coverages ranging from 1.6 to 4.2 μmol/m(2) (i.e., stationary phases yielding low to intermediate shape selectivity) in contact with a 67/33 mol% acetonitrile/water mobile phase. The simulations show that the stationary phase acts as a very heterogeneous environment where analytes with different shapes prefer different spatial regions with specific local bonding environments of the ODS chains. However, these favorable retentive regions cannot be described as pre-existing cavities because the chain conformation in these local stationary phase regions adapts to accommodate the analytes.

Retention prediction in reversed‐phase liquid chromatography systems with methanol/water mobile phases containing different alkanols as additives

In an effort to gain enhancement of selectivity in reversed-phase liquid chromatography, retention was tuned in this study by introducing short and medium straight-chained-length alkanol additives (methanol (MeOH), ethanol, 1-propanol, 1-butanol, 1-pentanol, 1-hexanol or 1-heptanol) at low concentrations in mobile phases containing MeOH as the main organic solvent. A six-parameter retention model considering simultaneously the contents of the main organic modifier and of the alcohol additive as well as of the number of alkyl chain of additive was developed by a direct combination of equations expressing separately a linear dependence of the retention upon each of these factors. The effectiveness of the above model was tested in the retention prediction of a mixture of six alkylbenzenes under isocratic conditions with mobile phases containing as an additive any member of the homologues series of alkanols (with 1-7 carbon atoms) at different low concentrations in a wide range of MeOH-water mixtures. The prediction was excellent in all cases even when the alkanol additives used in experiments for the fitting procedure are different than those used in chromatographic runs done for testing the prediction ability of the proposed model.

Utility of Retention Prediction Model for Investigation of Peptide Separation Selectivity in Reversed-Phase Liquid Chromatography: Impact of Concentration of Trifluoroacetic Acid, Column Temperature, Gradient Slope and Type of Stationary Phase

Peptide separation selectivity in reversed-phase liquid chromatography was investigated using a training set of 165 peptides with a total of 1698 amino acid residues. Gradient separation was performed at selected chromatographic conditions, varying column temperature, gradient slope, ion-pairing reagent concentration, and type of stationary phase. The retention times for each set of experiments were utilized to calculate the amino acid retention coefficients using a published prediction model (Rapid Commun. Mass Spectrom. 2007, 21, 2813-2821). The calculated retention coefficients reflect the contribution of each type of amino acid residue to peptide retention at a given chromatographic condition. For example, the concentration of ion-pairing reagent (trifluoroacetic acid) had the strongest impact on the retention of peptides containing an increasing number of basic (charged) amino acids, such as arginine, lysine, and histidine, while the retention coefficients of other amino acids are minimally affected. Increasing the separation temperature resulted in a moderate decrease of amino acid retention coefficients with the exception of isoleucine, leucine, valine, and proline. This finding suggests that these residues enhance peptide retention at elevated temperature. Amino acid residue retention coefficients were also helpful to understand the impact of sorbent pore size (130 vs 300 A) on peptide retention selectivity. In addition we investigated the selectivity differences of various reversed-phase chromatographic sorbents. The trends suggested by calculated retention coefficients were confirmed using a set of synthetic peptides with specifically designed sequences.

Effect of temperature on the chromatographic retention of ionizable compounds: II. Acetonitrile–water mobile phases

The retentive behavior of weak acids and bases in reversed-phase liquid chromatography (RPLC) upon changes in column temperature has been theoretically and experimentally studied. The study focuses on examining the temperature dependence of the retention of various solutes at eluent pH close to their corresponding p K a values, and on the indirect role exerted by the buffer ionization equilibria on retention and selectivity. Retention factors of several ionizable compounds in a typical octadecylsilica column and using buffer solutions dissolved in 30% (v/v) acetonitrile as eluent at five temperatures in the range from 25 to 50 °C were carefully measured. Six buffer solutions were prepared from judiciously chosen conjugated pairs of different chemical nature. Their p K a values in this acetonitrile–water composition and within the range of 15–50 °C were determined potentiometrically. These compounds exhibit very different standard ionization enthalpies within this temperature range. Thus, whenever they are used to control mobile phase pH, the column temperature determines their final pH. Predictive equations of retention that take into account the temperature effect on both the transfer and the ionization processes are evaluated. This study demonstrates the significant role that the selected buffer would have on retention and selectivity in RPLC at temperatures higher than 25 °C, particularly for solutes that coelute.

Column selectivity in reversed-phase liquid chromatography

As reported previously, five solute-column interactions (hydrophobicity, steric resistance, hydrogen-bond acidity and basicity, ionic interaction) quantitatively describe column selectivity for 163 alkyl-silica, polar-group and cyano columns. In the present study, solute retention and column selectivity for 11 phenyl and 5 fluoro-substituted columns were compared with alkyl-silica columns of similar ligand length. It is concluded that two additional solute-column interactions may be significant in affecting retention and selectivity for the latter columns: (a) dispersion interactions of varying strength as a result of significant differences in bonded-phase polarizability or refractive index and (b) pi-pi interactions in the case of phenyl columns and aromatic solutes. These 16 phenyl and fluoro columns were also characterized in terms of hydrophobicity, steric resistance, hydrogen-bond acidity and basicity, and ionic interaction.

Column selectivity in reversed-phase liquid chromatography

Eleven cyanopropyl ("cyano") columns were characterized by means of a relationship developed originally for alkyl-silica columns. Compared to type-B alkyl-silica columns (i.e., made from pure silica), cyano columns are much less hydrophobic (smaller H), less sterically restricted (smaller S*), and have lower hydrogen-bond acidity (smaller A). Because sample retention is generally much weaker on cyano versus other columns (e.g., C8, C18), a change to a cyano column usually requires a significantly weaker mobile phase in order to maintain comparable values of k for both columns. For this reason, practical comparisons of selectivity between cyano and other columns (i.e., involving different mobile phases for each column) must take into account possible changes in separation due to the change in mobile phase, as well as change in the column.

Effect of temperature on the chromatographic retention of ionizable compounds: I. Methanol–water mobile phases

The retention mechanism of acids and bases in reversed-phase liquid chromatography (RPLC) has been experimentally studied by examining the temperature dependence of retention, with emphasis on the role of the buffer ionization equilibria in the retention and selectivity. Retention factors of several ionizable compounds in a typical octadecylsilica column and using buffers dissolved in 50% (w/w) methanol as eluents at three temperatures in the range of 25–50 °C were measured. Two pairs of buffer solutions were prepared by a close adjusting of their pH at 25 °C; differences in their ionization enthalpies determined a different degree of ionization when temperature was raised and, as a consequence, a different shift in the eluent pH. Predictive equations of retention that take into account the temperature effect on both the transfer and the ionization processes are proposed. This study demonstrates the significant role that the selected buffer would have in retention and selectivity in RPLC at temperatures higher than 25 °C, particularly for co-eluted solutes.

Simulation Studies on the Effects of Mobile-Phase Modification on Partitioning in Liquid Chromatography

Various driving forces have been suggested to explain retention and selectivity in reversed-phase liquid chromatography (RPLC). To provide molecular-level information on the retention mechanism in RPLC, configurational-bias Monte Carlo simulations in the Gibbs ensemble were carried out for model systems consisting of three phases: an n-hexadecane retentive phase, a mobile phase with varying water-methanol composition, and a helium vapor phase as reference state. Liquid n-hexadecane functions as a model of a hydrophobic stationary phase, and a wealth of experimental data exists for this system. Gibbs free energies for solute transfers from gas to retentive phase, from gas to mobile phase, and from mobile to retentive phase were determined for a series of short linear alkanes and primary alcohols. Although the magnitude of the incremental Gibbs free energy of transfer for a methylene segment is always larger for the gas- to retentive-phase transfer than the gas- to mobile-phase transfer, it is found that the partitioning of alkanes and alkyl tail groups is mostly affected by the changes in the aqueous mobile phase that occur when methanol modifiers are added. In contrast, the partitioning of the alcohol headgroup is sensitive to changes in both the n-hexadecane and the mobile phases. In particular, it is found that hydrogen-bonded aggregates of methanol are present in the n-hexadecane phase for higher methanol concentrations in the mobile phase. These aggregates strongly increase alcohol partitioning into the retentive phase. The simulation data clearly demonstrate that due to modification of the retentive-phase hydrocarbons by solvent components, neither the solvophobic theory of RPLC, advocated by Horvath and co-workers, nor the lipophilic theory of RPLC, advocated by Carr and co-workers, can adequately describe the separation mechanism of the hexadecane model system of a retentive phase studied here nor the more complex situation present in actual RPLC systems.

Column selectivity in reversed-phase liquid chromatography: VI. Columns with embedded or end-capping polar groups

A previous model of column selectivity for reversed-phase liquid chromatography (RP-LC) has been applied to an additional 21 columns with embedded or end-capping polar groups (EPGs). Embedded-polar-group columns exhibit a significantly different selectivity vs. non-EPG, type-B columns, generally showing preferential retention of hydrogen-bond donors, as well as decreased retention for hydrogen-bond acceptors or ionized bases. EPG-columns are also generally less hydrophobic (more polar) than are non-EPG-columns. Interestingly, columns with polar end-capping tend to more closely resemble non-EPG columns, suggesting that the polar group has less effect on column selectivity when used to end-cap the column versus the case of an embedded polar group. Column selectivity data reported here for EPG-columns can be combined with previously reported values for non-EPG columns to provide a database of 154 different columns. This enables a comparison of any two of these columns in terms of selectivity. However, comparisons that involve EPG columns are more approximate.

Chemometric studies of polycyclic aromatic hydrocarbon shape selectivity in reversed-phase liquid chromatography.

The molecular shape recognition differences between monomeric and polymeric C18 stationary phases in the reversed-phase liquid chromatography (RPLC) separation of unsubstituted polycyclic aromatic hydrocarbons (PAHs) and methyl-substituted polycyclic aromatic hydrocarbons (MPAHs) are examined through the use of partial least squares (PLS) analysis techniques. The resulting PLS models are able to describe the enhanced shape selectivity of the polymeric phase for recognizing subtle structural differences among planar and nonplanar isomers. PLS component analyses of these models reveal that spatial and topological descriptors are primarily used to rank structural differences among the PAHs (i.e., fused-ring patterns, molecular length and breadth) that control such shape-selective chromatographic processes. This is consistent with the view that polymeric alkyl chain stationary phases contain size- and shape-specific "slots" that promote the separation of structurally-related solutes. In contrast, the monomeric phase model is limited in resolving both the isomer classes and the nonplanarity shape differences among the PAHs. However, an improvement of shape recognition on the monomeric phase was elucidated by the PLS model for two PAHs (phenanthro[3,4-c]phenanthrene and dibenzo[g,p]chrysene) exhibiting the most extreme nonplanarity. These results suggest that a limited amount of space between alkyl chains may exist within the higher-density polymeric phase to recognize shape differences among the bulkier and nonplanar solutes.

Column selectivity in reversed-phase liquid chromatography: I. A general quantitative relationship

Retention factors k have been measured for 67 neutral, acidic and basic solutes of highly diverse molecular structure (size, shape, polarity, hydrogen bonding, p K a, etc.) on 10 different C 18 columns (other conditions constant). These data have been combined with k values from a previous study (86 solutes, five different C 8 and C 18 columns) to develop a six-term equation for the correlation of retention as a function of solute and column. Values of k can be correlated with an accuracy of ±1–2% (1 standard deviation). This suggests that all significant contributions to column selectivity have been identified (and can be measured) for individual alkyl-silica columns which do not have an embedded polar group. That is, columns of the latter kind can be quantitatively characterized in terms of selectivity for use in the separation of any sample.

Column selectivity in reversed-phase liquid chromatography: III. The physico-chemical basis of selectivity

Reversed-phase liquid chromatography (RP-LC) retention data for 23 additional solutes have been acquired to further test and evaluate a general relationship from part I: (1) log α= log (k/k ref )= η′ H (i) + σ′ S (ii) + β′ A (iii) + α′ B (iv) + κ′ C (v) The physico-chemical origin of terms i– v above is examined here by comparing values of (a) the solute parameters of Eq. (1) ( η′, σ′, etc.) vs. solute molecular structure, and (b) the column parameters ( H, S, etc.) vs. column properties (ligand length and concentration, pore diameter, end-capping). We conclude that terms i– v correspond, respectively, to hydrophobic ( i), steric ( ii), hydrogen bonding ( iii, iv) and ionic ( v) interactions between solute and stationary phase. While steric interaction (term ii) is superficially similar to what previously has been defined as “shape selectivity”, the role of the solute and column in determining steric selectivity (term ii) appears more complex than previously proposed for “shape selectivity”. Similarly, what has previously been called hydrogen bonding between donor solutes and an acceptor group in the stationary phase (term iv) is very likely an oversimplification.

Column selectivity in reversed-phase liquid chromatography: II. Effect of a change in conditions

The isocratic retention of 67 widely-different solutes in reversed-phase liquid chromatography (RP-LC) has been investigated as a function of temperature and mobile phase composition (% B) for three different C 18 columns. Similar studies were also carried out in a gradient mode, where temperature, gradient time and solvent type were varied. These results show that changes in retention with these conditions are similar for each of these three columns. This suggests that relative column selectivity as defined by experiments for one set of experimental conditions will be approximately applicable for other conditions, with the exception of changes in mobile phase pH—which can affect values of the column parameter C (a measure of silanol ionization). Column selectivity as a function of pH was explored for several columns.

Effect of molecular interactions on retention and selectivity in reversed-phase liquid chromatography

The linear solvation energy relationships (LSERs) have been applied in the last years for description and prediction of retention and selectivity in reversed-phase liquid chromatography with good results. Widely different stationary phases have been compared and characterized by LSERs. In recent publications the influence of the type of the organic moderator and the composition of the mobile phase have also been described. However, the influence of the molecular properties of the solutes to be separated has never been discussed. According to the LSER model variation in retention factors (log k) with solute structure can be related to their potential for various intermolecular interactions. The retention factor is given as the sum of the terms of the LSER equation representing various types of molecular interactions. For this reason the influence of the structure and molecular properties of the solutes to be separated can also be investigated using the LSER equation. In this study we shall demonstrate how the specific molecular interactions influence chromatographic retention and selectivity. We intend to show that retention and selectivity depend on all participants of the system. In addition to the structure and properties of the stationary phase and the type and composition of the mobile phase the molecular properties of the solutes, characterized by the solvation parameters, will also influence the type and extent of the various molecular interactions governing retention and selectivity.

An attempt directed toward enhanced shape selectivity in reversed-phase liquid chromatography: preparation of the dodecylaminated beta-cyclodextrin-bonded phase.

With the aim of preparing a stationary phase with a high shape-recognition ability for liquid chromatography, a new bonded phase was synthesized by coupling multiply dodecylamino-substituted beta-cyclodextrin (beta-CD) to 3-glycidoxypropyl-derivatized silica gel. The stationary phase prepared in this way was expected to have increased shape selectivity compared with that of conventional reversed-phase materials, due to solute interactions with the alkyl chain piles built up on the beta-CDs bonded to silica. The separation characteristics of the bonded phase were investigated using polycyclic aromatic hydrocarbons (PAHs) with different molecular shapes and compared with those of monomeric ODS and native beta-cyclodextrin-bonded phases. The newly developed stationary phase was found to be highly selective for PAHs.

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 C 18 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.