Hydrophobic Subtraction Model Research Articles

A simulation-guided approach to the selection of RPLC columns for platform small molecule separations

Simulations were conducted to evaluate the potential of several hundred reversed-phase columns to separate small molecules. By calculating the retention factor of compounds in randomly generated virtual mixtures via the HSM (hydrophobic subtraction model) and applying basic chromatography theory, the simulation can estimate the retention time and peak width of every virtual compound and calculate the resolution between every adjacent pair of compounds. A preferred column set based on the number of successful separations of randomly generated virtual mixtures was developed. The tandem-column liquid chromatography (TC-LC) approach can separate 53.2 % of the 16-compound samples using 20 tandem-column pairs, while a single-column approach can only separate 42.6 % of the 16-compound samples with 20 single columns. The preferred set of columns obtained from the simulation was almost the same as the empirical set of columns previously obtained. In screening applications, TC-LC can achieve a comparably successful separation factor (selectivity) with a smaller column inventory (nine 50-mm columns) compared to the larger inventory needed by single-column LC (twenty-one 100-mm columns).

In-silico method development and optimization of on-line comprehensive two-dimensional liquid chromatography via a shortcut model

Comprehensive two-dimensional liquid chromatography (LCxLC) represents a valuable alternative to conventional single column, or one-dimensional, liquid chromatography (1D-LC) for resolving multiple components in a complex mixture in a short time. However, developing LCxLC methods with trial-and-error experiments is challenging and time-consuming, which is why the technique is not dominant despite its significant potential. This work presents a novel shortcut model to in-silico predicting retention time and peak width within an RPLCxRPLC separation system (i.e., LCxLC systems that use reversed-phase columns (RPLC) in both separation dimensions). Our computationally effective model uses the hydrophobic-subtraction model (HSM) to predict retention and considers limitations due to the sample volume, undersampling and the maximum pressure drop. The shortcut model is used in a two-step strategy for sample-dependent optimization of RPLCxRPLC separation systems. In the first step, the Kendall's correlation coefficient of all possible combinations of available columns is evaluated, and the best column pair is selected accordingly. In the second step, the optimal values of design variables, flow rate, pH and sample loop volume, are obtained via multi-objective stochastic optimization. The strategy is applied to method development for the separation of 8, 12 and 16 component mixtures. It is shown that the proposed strategy provides an easy way to accelerate method development for full-comprehensive 2D-LC systems as it does not require any experimental campaign and an entire optimization run can take less than two minutes.

Mechanistic studies to understand peak tailing due to sulfinic acid- and carboxylic acid-silanophilic interactions in reversed-phase liquid chromatography

Silanophilic interactions are a primary contributor to peak tailing of acidic pharmaceutical compounds, thus a thorough understanding is especially important for reversed-phase liquid chromatography (RPLC) method development. Herein, a sulfinic acid compound that exhibited severe peak tailing in RPLC with acidic mobile phases was carefully studied. Results indicated that the neutral protonated form of the sulfinic acid is involved in the strong interaction that leads to peak tailing, but that tailing can be mitigated with a blocking effect achieved through use of acetic acid modifier in the mobile phase. Peak tailing was also observed with other structurally-similar sulfinic acids and carboxylic acids but was, in general, less severe with the latter. The Hydrophobic Subtraction Model (HSM) was applied to six commercial C18 columns that exhibited different tailing behaviors for the sulfinic acid compound in attempts to identify key sites of interaction within the stationary phase. A combination of heated acid column wash experiments and density functional theory (DFT) calculations indicate that the differential interactions of the acids with vicinal silanol pairs in the stationary phase play a major role in peak tailing.

Changes in the cis-trans isomer selectivity of a reversed-phase liquid chromatography column during use with acidic mobile phase conditions

Reversed-phase liquid chromatography (RPLC) is the analytical tool of choice for monitoring process-related organic impurities and degradants in pharmaceutical materials. Its popularity is due to its general ease-of-use, high performance, and reproducibility in most cases, all of which have improved as the technique has matured over the past few decades. Nevertheless, in our work we still occasionally observe situations where RPLC methods are not as robust as we would like them to be in practice due to variations in stationary phase chemistry between manufactured batches (i.e., lot-to-lot variability), and changes in stationary phase chemistry over time. Over the last three decades several models of RPLC selectivity have been developed and used to quantify and rationalize the effects of numerous parameters (e.g., effect of bonded phase density) on separation selectivity. The Hydrophobic Subtraction Model (HSM) of RPLC selectivity has been used extensively for these purposes; currently the publicly available database of column parameters contains data for 750 columns. In this work we explored the possibility that the HSM could be used to better understand the chemical basis of observed differences in stationary phase selectivity when they occur – for example, lot-to-lot variations or changes in selectivity during column use. We focused our attention on differences and changes in the observed selectivity for a pair of cis-trans isomers of a pharmaceutical intermediate. Although this is admittedly a challenging case, we find that the observed changes in selectivity are not strongly correlated with HSM column parameters, suggesting that there is a gap in the information provided by the HSM with respect to cis-trans isomer selectivity specifically. Further work with additional probe molecules showed that larger changes in cis-trans isomer selectivity were observed for pairs of molecules with greater molecular complexity, compared to the selectivity changes observed for simpler molecules. These results do not provide definitive answers to questions about the chemical basis of changes in stationary phase chemistry that lead to observed differences in cis-trans isomer selectivity. However, the results do provide important insights about the critical importance of molecular complexity when choosing probe compounds and indicate opportunities to develop improved selectivity models with increased sensitivity for cis-trans isomer selectivity.

Development of tandem-column liquid chromatographic methods for pharmaceutical compounds using simulations based on hydrophobic subtraction model parameters

The liquid chromatography (LC) analysis of small molecule pharmaceutical compounds and related impurities is crucial in the development of new drug substances, but developing these separations is usually challenging due to analyte structural similarities. Tandem-column LC (TC-LC) has emerged as a powerful approach to achieve alternative separation selectivity compared to conventional single column separations. However, one of the bottlenecks associated with use of tandem column approaches is time-consuming column pair screening and selection. Herein, we compared critical resolution (Rc) in single column vs. TC-LC separations for a given set of small molecule pharmaceutical compounds and developed a column selection workflow that uses separation simulations based on parameters from the hydrophobic subtraction model (HSM) of reversed-phase selectivity.In this study, HSM solute parameters were experimentally determined for a small molecule pharmaceutical (Linrodostat) and ten of its related impurities using multiple linear regression of their retentions on 16 selected RPLC columns against in-house determined HSM column parameters. Rc values were calculated based on HSM database column parameters for a pool of about 200 available stationary phases in both single-phase column (2.1 mm i.d. × 100 mm) or tandem column paired (two 2.1 mm i.d. × 50 mm) formats. Four column configurations (two single and two tandem) were predicted to achieve successful separations under isocratic HSM separation conditions, with a fifth tandem pair predicted to have a single co-elution. Of these five potential candidates, one tandem pair yielded compete baseline resolution of the 11-component mixture in an experimental separation.In this specific case, the tandem column pairs outperformed single-phase columns, with better predicted and experimental Rc values for the Linrodostat mixture under the HSM separation conditions. The results reported in this study demonstrated the enormous selectivity potential of TC-LC in pharmaceutical compound separations and are consistent with our previous study that examined the potential of tandem column approaches using purely computational means, though there is room for substantial improvement in the prediction accuracy. The proposed workflow can be used to prioritize a small number of column combinations by computational means before any experiments are conducted. This is highly attractive from the point of view of time and resource savings considering over 200,000 different tandem column pairings are possible using columns for which there are data in the HSM database.

Direct electrochemical monitoring of in vitro glucuronidation metabolism by the hydrophobic selectivity of lipophilic micelles

Glucuronidation metabolism plays an important role in the detoxification and clearance of lipophilic drugs. High-performance liquid chromatography (HPLC) is the most common technology for the separation and analysis of drug metabolism. Inspired by the hydrophobic selectivity of the alkyl bonded silica stationary phase in HPLC, we prepared the functional membrane consisting of lipophilic micelles and mesoporous silica (designated as LM@MSM) modified on indium tin oxide (ITO) electrode, and performed a detailed investigation of its permselectivity towards different anthraquinones (AQs) and their glucuronide metabolites. We demonstrated that the diffusion flux of these AQ analytes was relevant to their chemical structure and partition coefficient, thus leading to the hydrophobic selectivity of the LM@MSM. This selectivity was further supported by the retention behavior of these AQ analytes in HPLC based on the hydrophobic-subtraction model. Consequently, the developed LM@MSM sensor was successfully applied to the direct and rapid electrochemical monitoring of in vitro glucuronidation metabolism of lipophilic AQs with high selectivity and sensitivity.

Separation of Panax notoginseng saponins on modified rosin ester-bonded silica stationary phase and its mechanism

A column prepared using a unique three-membered phenanthrene skeleton of rosin has complementary selectivity to a C18 column for some separation tasks. In this study, propylene pimaric acid (16-hydroxyethyl acrylate-34-n-butyl) ester (BRB) and propylene pimaric acid (16-hydroxyethyl acrylate-34-dodecyl) ester (BRLA) were used as functional ligands to prepare two novel stationary phases, namely BRB@SiO2 and BRLA@SiO2, through a “thiol-ene” click chemistry reaction. The characterization results of Fourier transform infrared spectroscopy, thermogravimetric analysis, nitrogen adsorption-desorption measurements, and contact angle tests showed that the BRB@SiO2 and BRLA@SiO2 stationary phases were successfully prepared. In addition, the performance of the columns was evaluated using the Tanaka test and hydrophobic subtraction model, which showed that the stationary phases exhibited typical reversed-phase chromatography performance and good hydrophobicity, hydrophobic selectivity, and steric selectivity. The changes in the retention of Panax notoginseng saponins on a column under different chromatographic conditions (acetonitrile content, flow rate, and column temperature) were investigated. The separation effect of BRB@SiO2 and BRLA@SiO2 columns on P. notoginseng saponins was better than that of the C18 column and the BRLA@SiO2 column could replace the C18 column for the detection of P. notoginseng saponins.

Are two liquid chromatography columns in tandem better than one?: Answers from the hydrophobic subtraction model

An approach is described for determining if there is an intrinsic advantage, from a selectivity and resolution perspective, of using two different UHPLC/HPLC reversed-phase columns in tandem for a separation of a given sample compared to a single U/HPLC reversed-phase column that provides the same plate number. Retention data for 16 compounds extracted directly from the hydrophobic subtraction model (HSM) database at HPLCColumns.org are used to simulate and then compare the critical resolution of those compounds obtained using HSM conditions (isocratic elution at 35°C using 50% acetonitrile, 50% aqueous phosphate buffer at pH 2.8 or 7) for each of 662 U/HPLC single columns or 218,791 combinations of tandem columns and assuming a modest plate number of 8000. The critical resolution obtained for 16 additional “n-1” samples created by the systematic removal of one of the original 16 compounds was also compared using single- and tandem-column LC, as was the critical resolution obtained for thousands of synthetic samples generated by randomly varying HSM solute descriptors for each synthetic compound. When all possible single-column or tandem-column results were compared, a significant advantage was observed with tandem-column liquid chromatography (TC-LC), with an average increase in critical resolution of 0.63 (pH 2.8) or 0.75 (pH 7) units observed for the synthetic samples with the smallest number of components (m = 5). As the number of components in a sample increased, the average improvement in critical resolution (∆Rs,crit) using TC-LC gradually decreased from about 0.70 for m = 5 to 0.18 for m = 32 components. The average improvement in critical resolution achieved by switching from SC-LC to TC-LC was also lower when a smaller number of columns and column combinations were available to explore, as would be the case for a finite column inventory in a real laboratory. Nevertheless, on average there does appear to be an intrinsic advantage of tandem-column liquid chromatography, however small, which can be amplified by using high efficiency columns.

Investigation of stationary phases performance for eicosanoids profiling in RP-HPLC.

Eicosanoids - oxidative derivatives from arachidonic acid - represent biologically active lipid mediators in inflammatory processes. Different analytical methods treat eicosanoid analysis. Among which, reverse phase liquid chromatography figures as the appropriate method for eicosanoid profiling. RP-HPLC for eicosanoid analysis is often conducted on C18 columns. Some studies focused on profiling one family of eicosanoids; others considered all eicosanoid families. In both cases, co-elution remained a major issue and detection in mass spectrometry partially resolves this problem. In fact, the mass transitions used to monitor eicosanoid species are not specific enough and many isobars can be listed. For this, optimizing the RP-HPLC separation remains important. Based on the parameter Fs - deriving from the hydrophobic-subtraction model - and radar plots, we chose columns with different selectivities. The hydrophobic-subtraction model guided our interpretation of molecular interactions between eicosanoids and stationary phases. We founded our approach for selectivity optimization on peak capacity per minute and time needed values. Herein, we screened seven stationary phases and evaluated their chromatographic performances in RP-HPLC. Stationary phases presented different chemistry, type of silica, length, and particle size. Superficially porous particle columns registered better chromatographic profiles than classical stationary phases; and columns with embedded polar group did not serve our purpose. The stationary phase Accucore C30 - even being the least retentive - revealed the best selectivity and efficiency, and recorded the shorter duration for eicosanoid analysis.

Improvements in the predictive accuracy of the hydrophobic subtraction model of reversed-phase selectivity

The hydrophobic subtraction model (HSM) for characterizing the selectivity of reversed-phase liquid chromatography (LC) columns has been used extensively by the LC community since it was first developed in 2002. Continuing interest in the model is due in part to the large, publicly available set of column descriptors that has been assembled over the past 18 years. In the work described in this report, we sought to refine the HSM with the goal of improving the predictive accuracy of the model without compromising its physico-chemical interpretability. The approach taken here has the following facets. A set of retention measurements for 635 columns and the 16 probe solutes used to characterize new columns using the HSM was assembled. Principal components analysis (PCA) was used as a guide for the development of a refined version of the HSM. Several outlying columns (84) were eliminated from the analysis because they were either inconsistent with the PCA model or were outliers from the original HSM model. With the retention dataset for the 16 probe solutes on the remaining 551 columns, we determined that a six-component model is the most sophisticated form of the model that can be used without overfitting the data. In our refined version of the HSM, the S*σ term has been removed. Two new terms have been added, which more accurately account for the molecular volume of the solute (Vv), and the solute dipolarity (Dd), and the remaining terms have been adjusted to accommodate these changes. The refined model described here provides improved prediction of retention factors, with the model standard error being reduced from 1.0 for the original HSM to 0.35 for the refined model (16 solutes, 551 columns). Furthermore, the number of retention factors with errors greater than 10% are reduced from 231 to 25. A revised metric for column similarity, F, is also proposed as a part of this work.

Investigation into reversed phase chromatography peptide separation systems part III: Establishing a column characterisation database

The Peptide RPC Column Characterisation Protocol was applied to 38 stationary phases, varying in ligand chemistry, base silica, end capping and pore size, which are suitable for the analysis of peptides. The protocol at low and intermediate pH is based on measuring retention time differences between peptides of different functionality to calculate selectivity delta values. The characterisation was designed to explore increases / decreases in positive or negative charge (deamidation), steric effect (i.e. racemisation / switch in amino acid order), oxidation and addition / removal of aromatic moieties. The necessity of developing a characterisation protocol specifically for peptide analysis was highlighted by the fact that the small molecule databases (Snyder's Hydrophobic Subtraction Model and the extended Tanaka protocol) failed to correlate with the Peptide RPC Column Characterisation Protocol. Principal Component Analysis was used to demonstrate that the protocol could be used to identify columns with similar or dissimilar chromatographic selectivity for the purpose of selectivity back-up or method development columns respectively. This was validated using peptide fragments derived from the tryptic digest of bovine insulin and carbonic anhydrase. It was also demonstrated that the presence of positively charged functional groups on the stationary phase was advantageous as it yielded very different chromatographic selectivity and improved peak shape.

Localised quantitative structure–retention relationship modelling for rapid method development in reversed-phase high performance liquid chromatography

Quantitative structure–retention relationships (QSRR) predicting the values of solute “hydrophobicity” coefficient η′ in the approximate hydrophobic subtraction model (HSM) can be used to predict retention times of compounds on numerous reversed-phase (RP) columns, provided that column parameters on the corresponding stationary phases are available. In the present study, we propose a new dual clustering-based localised QSRR approach, combining P-ratio clustering (where P is the octanol–water partition coefficient) with second dominant interaction (SDI)-based clustering, to produce predictive models with an acceptable level of prediction accuracy for in silico column scoping in RP method development. QSRR models for η′ values were derived for 49 compounds out of 63 in a dataset extracted from the literature, where retention data were measured under one isocratic mobile phase condition (i.e., acetonitrile-water, 50:50 [v/v]). These models gave a predictive squared correlation coefficient Qext(F2)2 of 0.83 and a root mean square error of prediction (RMSEP) of 0.14. For the modelling, a genetic algorithm-partial least square regression (GA-PLS) approach was performed using the η′ values and their relevant molecular descriptors. The corresponding retention times were predicted by applying the predicted η′ values of the models and the stationary phase “hydrophobicity” parameter H values for the corresponding columns to the approximate HSM, resulting in excellent accuracy and predictability (Qext(F2)2 of 0.90 and RMSEP of 0.72 min). The established QSRR approach was experimentally verified for six Thermo Scientific columns (Acclaim™ 120 C18, Acclaim Polar Advantage, Acclaim Polar Advantage II, Accucore™ aQ, Accucore Phenyl-X, and Hypersil Gold C18 columns) using two types of datasets. The first dataset consisted of eight model compounds extracted from the original dataset and retention time predictions for those compounds were then evaluated on the above columns. The result showed good agreement between predicted and observed retention times with an acceptable error in retention time predictions (slope of 0.97, Qext(F2)2 of 0.95, a mean absolute error (MAE) of 0.43 min and RMSEP of 0.61 min). The second dataset included eight test compounds not included in the original dataset, which were all classified into the η′ cluster by applying a Tanimoto similarity (TS) threshold of 0.7. Similarly, predicted retention times of the test compounds were compared with their corresponding observed retention times, resulting in acceptable retention time predictions with the slope of 0.99, Qext(F2)2 of 0.93 and RMSEP of 0.52 min. Comparisons of resolution values between columns were utilised to select the most suitable columns for separations of the compounds in the respective test sets. Actual chromatograms obtained on the chosen columns showed the feasibility for effective column scoping without experimentation on numerous RP stationary phases available in the USP website, based on the predicted resolution values.

Inside Back cover: Retention prediction using quantitative structure‐retention relationships combined with the hydrophobic subtraction model in reversed‐phase liquid chromatography

Inside Back cover: Retention prediction using quantitative structure‐retention relationships combined with the hydrophobic subtraction model in reversed‐phase liquid chromatography

Retention prediction using quantitative structure-retention relationships combined with the hydrophobic subtraction model in reversed-phase liquid chromatography.

The hydrophobic subtraction model (HSM) combined with quantitative structure-retention relationships (QSRR) methodology was utilized to predict retention times in reversed-phase liquid chromatography (RPLC). A selection of new analytes and new RPLC columns that had never been used in the QSRR modeling process were used to verify the proposed approach. This work is designed to facilitate early prediction of co-elution of analytes in pharmaceutical drug discovery applications where it is advantageous to predict whether impurities might be co-eluted with the active drug component. The QSRR models were constructed through partial least squares regression combined with a genetic algorithm (GA-PLS) which was employed as a feature selection method to choose the most informative molecular descriptors calculated using VolSurf+ software. The analyte hydrophobicity coefficient of the HSM was predicted for subsequent calculation of retention. Clustering approaches based on the local compound type and the local second dominant interaction were investigated to select the most appropriate training set of analytes from a larger database. Predicted retention times of five new compounds on five new RPLC C18 columns were compared with their measured retention times with percentage root-mean-square errors of 15.4 and 24.7 for the local compound type and local second dominant interaction clustering methods, respectively.

Reversed-phase liquid chromatography system constant database over an extended mobile phase composition range for 25 siloxane-bonded silica-based columns

The solvation parameter model is used to characterize the retention properties of 25 siloxane-bonded type-B silica columns under typical reversed-phase liquid chromatography conditions with binary aqueous mobile phases containing 10–70% (v/v) methanol and acetonitrile. To further explore the affect of solvent type on column selectivity complementary information is presented for aqueous phases containing 10–70% (v/v) tetrahydrofuran for two columns. Columns were selected to include examples of all common column packing morphologies for small molecule separations (totally porous particles, superficially porous particles, organic–inorganic hybrid particles, and a monolith) and common topologies octadecylsiloxane-bonded (including phases with a polar-embedded group, mixed-mode phases with a short-chain siloxane-bonded polar functional group, sterically crowded, positive shield, and fluorine-containing phases); octylsiloxane-bonded; and various phenyl-containing stationary phases with different linker arms, pentafluorophenylalkyl and biphenyl groups. The column properties are reported as a system constant database and as system maps for the full range of mobile phase compositions. Selectivity differences are evaluated using principal component factor analysis to classify the columns into selectivity groups and correlation plots of the system constants for compared columns at all mobile phase compositions. The later is shown to be a suitable technique to identify (near) selectivity equivalent columns and varied columns suitable for method development. Contributions to retention from steric resistance and cation-exchange interactions not parameterized in the solvation parameter model are identified for columns and conditions where they may be important. It is shown that the interaction parameters employed in the hydrophobic-subtraction model have little overlap with those of the solvation parameter model and a quantitative comparison of column properties delineated by both models is not possible.

A pH-stable, crosslinked stationary phase based on the thiol-yne reaction

Stationary phases that can withstand extremes of pH and temperature are needed to allow a single column to accommodate a wider set of solutes and separation criteria. We used a simple multi-step process using the thiol-yne reaction following the modification of the silica surface with a thiol-containing silane. The monomers 1,4-diethynylbenzene (DEB) and 1,6-hexanedithiol were used to create a crosslinked thiol-yne (CTY) stationary phase along the surface of the thiol functionalized silica. In the Tanaka test characterization, the CTY phase showed a low phase ratio, methylene selectivity typical of a reversed phase, and extremely high shape selectivity compared to commercial reversed phases. The hydrophobic subtraction model characterization showed a high positive steric resistance, a low hydrogen bond acidity, and a high cation-exchange capacity compared to most reversed phases. At pH 0.5 with an 85% aqueous mobile phase the phase showed no significant change over 114 h. With a 50% aqueous mobile phase the phase took four more days than a sterically protected C18 phase for the k’ to decline 25%. At pH 12.6, 50% aqueous mobile phase, a sterically protected C18 phase showed a 20% decrease in k’ and more than a 60% decrease in theoretical plates per meter in three hours. The CTY phase actually showed modest increases in k’ and theoretical plates per meter after three hours.

Column selection for comprehensive two-dimensional liquid chromatography using the hydrophobic subtraction model

Two-dimensional (2D) liquid chromatography (2DLC) methods have grown in popularity due to their enhanced peak capacity that allows for resolving complex samples. Given the large number of commercially available column types, one of the major challenges in implementing 2DLC methods is the selection of suitable column pairs. Column selection is typically informed by chemical intuition with subsequent experimental optimization. In this work a computational screening method for 2DLC is proposed whereby virtual 2D chromatograms are calculated utilizing the Snyder–Dolan hydrophobic subtraction model (HSM) for reversed-phase column selectivity. Towards this end, 319 225 column pairs resulting from the combination of 565 columns and 100 sets of 1000 diverse analytes are examined. Compared to other screening approaches, the present method is highly predictive for column pairs that are able to resolve the largest number of analytes. This approach shows a strong sensitivity to the choice of the second dimension column (having a shorter operating time) and a preference for those with embedded polar moieties, whereas a relatively weak preference for C18 and phenyl columns is found for the first dimension.

Σpider diagram: A universal and versatile approach for system comparison and classification. Part 2: Stationary phase properties

Many different sorts of bonded phase chemistries may be used in high-performance liquid chromatography (HPLC) in the reversed-phase mode: C8, C18 (type A or B), polar-embedded C18, phenyl, pentafluorophenyl, or cyanopropyl. To assess their retention and selectivity properties, chromatographic tests exist. The data obtained from these tests may be presented in three ways. First, simple classification diagrams may be plotted, when only two or three parameters are studied. Secondly, chemometric treatments such as principal component analyses (PCA) or hierarchical cluster analyses (HCA) may be computed, when at least 4 parameters are studied. These are sometimes uneasy to interpret. Thirdly, the “distance” between one column and a reference column may be estimated, through calculated ranking (F or CDF) or selectivity (s) factors.In this paper, another treatment type is applied to the data of Euerby (Tanaka test) and Snyder (Hydrophobic Subtraction Model), each of these tests having 6 parameters. This treatment produces a visual classification, called spider diagram. In the first part of this series, this type of classification was applied to the classification of solvents. A logical and easily comprehensible classification is obtained for the varied types of bonded phases, with a clear location, which can be related to the chromatographic properties. The comparison of these diagrams shows that the classification based on Snyder’s hydrophobic subtraction model discriminates the stationary phases more effectively than the one based on the Tanaka test.Finally, on the basis of the parameter relevance and in order to favour comparison between these two tests and a third one called the carotenoid test, simplified classification maps are proposed. For Tanaka test, the selected parameters are the pentylbenzene retention factor (hydrophobicity), the benzylamine/phenol separation factor at pH 7.6 (polar surface activity) and the triphenylene/ortho-terphenyl (shape selectivity) separation factor. For Snyder test, the parameters selected are the ethylbenzene retention factor (hydrophobicity) the C term at pH 7.0 (polar surface activity) and the S* term (steric electivity). The location of some stationary phases onto the maps and their rankings are compared and shown to agree well between the three tests.

Separation of 1,4-Dihydropyridine Derivative and Its Oxidized Form

Derivatives of 1,4-dihydropyridine (DHP) still play an important role in treatment of cardiovascular diseases. Typical degradation of the DHP ring is aromatization to pyridine ring which occurs both chemically and biochemically. It is, therefore, important to have a reliable and robust analytical method for separation of DHPs from their oxidized counterparts. Separation of closely-related substances possessing similar hydrophobicity, such as DHP and its oxidized form, can be challenging on conventional alkyl-bonded sorbents. In this study, an impact of reversed-phase (RP) liquid chromatography conditions on separation of the DHP/Ox pair has been investigated. Initially, a systematic study has been performed on 33 commercial RP columns with mobile phase acetonitrile/water for separation of foridone and its corresponding oxidized form. The retention and selectivity are discussed in view of the hydrophobic-subtraction model. Best separation was found replacing conventional C18 sorbents with ones containing an embedded polar group due to polar interactions. Similarly, application of cyano columns resulted in efficient separation of analytes. Organic modifier of mobile phase (methanol vs. acetonitrile) contributed significantly to separation of foridone from its oxidized counterpart. Separation of six chemically diverse DHPs from corresponding oxidized forms was studied on seven RP columns (traditional C18 sorbent, alkyl sorbent with polar embedded group, two different aromatic phases, pentafluorophenylpropyl sorbent and sorbent with straight chain perfluorohexyl ligand). Both acetonitrile and methanol were applied as organic modifier. It was found that application of alkyl sorbent with an embedded polar group (column Zorbax Bonus RP) or cyano sorbent (column ACE CN) yields clear separation of chemically diverse DHPs from their oxidized forms.

A four parameter optimization and troubleshooting of a RPLC – charged aerosol detection stability indicating method for determination of S-lysophosphatidylcholines in a phospholipid formulation

A four parameter optimization of a stability indicating method for non-chromophoric degradation products of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1-stearoyl-sn-glycero-3-phosphocholine and 2-stearoyl-sn-glycero-3-phosphocholine was achieved using a reverse phase liquid chromatography-charged aerosol detection (RPLC-CAD) technique. Using the hydrophobic subtraction model of selectivity, a core-shell, polar embedded RPLC column was selected followed by gradient-temperature optimization, resulting in ideal relative peak placements for a robust, stability indicating separation. The CAD instrument parameters, power function value (PFV) and evaporator temperature were optimized for lysophosphatidylcholines to give UV absorbance detector-like linearity performance within a defined concentration range. The two lysophosphatidylcholines gave the same response factor in the selected conditions. System specific power function values needed to be set for the two RPLC-CAD instruments used. A custom flow-divert profile, sending only a portion of the column effluent to the detector, was necessary to mitigate detector response drifting effects. The importance of the PFV optimization for each instrument of identical build and how to overcome recovery issues brought on by the matrix effects from the lipid–RP stationary phase interaction is reported.