Linear Solvent Strength Model Research Articles

Linear solvent strength model on porous graphitic carbon stationary phase using high temperature liquid chromatographic method for allopurinol related substances analysis

A high-performance liquid chromatography (HPLC) method was developed for the analysis of Allopurinol and its Ph.Eur. impurities using a porous graphitic carbon (PGC) stationary phase. Retention behavior of solutes was studied across a wide temperature range (30–90 °C) and various gradient times (5–20 min). Analysis of the data revealed distinct retention mechanisms between reversed-phase and PGC phases. However, it was proved that the retention of Allopurinol and its Ph.Eur. impurities on PGC stationary phase can be effectively modeled using the linear solvent strength (LSS) theory. This allows for the utilization of LSS-based method development software to optimize methods under these conditions. By using commercial chromatographic modeling software, separation of Allopurinol and Ph.Eur. impurities was optimized within a large design space. At the optimized operating conditions (pH = 2.0, tG = 6 min, T = 60 °C), all solutes were separated within 6 min with baseline resolution. Comparison between predicted and experimentally measured chromatograms further confirmed the applicability of LSS theory in developing analytical methods for PGC-based HPLC systems. The presented approach offers a general framework for method development on PGC phases.

High-Throughput Chromatographic Separation of Oligonucleotides: A Proof of Concept Using Ultra-Short Columns.

Ion-pairing reversed-phase liquid chromatography (IP-RPLC) is the reference separation technique for characterizing oligonucleotides (ONs) and their related impurities. The aim of this study was to better understand the retention mechanism of ONs, evaluate the applicability of the linear solvent strength (LSS) retention model, and explore the potential of ultra-short columns having a length of only 5 mm for the separation of model ONs. First, the validity of the LSS model was evaluated for ONs having sizes comprised between 3 and 30 kDa, and the accuracy of retention time predictions was assessed. It was found that ONs in IP-RPLC conditions follow an "on-off" elution behavior, despite a molecular weight lower than that of proteins. For most linear gradient separation conditions, a column length between 5 and 35 mm was found to be appropriate. Ultra-short columns of only 5 mm were therefore explored to speed up separations by considering the impact of the instrumentation on the efficiency. Interestingly, the impacts of injection volume and post-column connection tubing on peak capacity were found to be negligible. Finally, it was demonstrated that longer columns would not improve selectivity or separation efficiency, but baseline separation of three model ONs mixtures was enabled in as little as 30 s on the 5 mm column. This proof-of-concept work paves the way for future investigations using more complex therapeutic ONs and their related impurities.

Retention prediction of monoamine neurotransmitters in gradient liquid chromatography.

Retention prediction of monoamine neurotransmitters has been compared for the generally applied linear solvent-strength model and quadratic polynomial three-parameter model. The design of experiments protocol has been applied to plan linear gradients within the experimental space with altered gradient time, mobile phase flow rate, and column temperature. Relative prediction errors increased at elevated temperature, which is more significant for the linear solvent-strength model when compared to the polynomial model. On the other hand, the predefined design of experiments space controls the retention time errors, as predictions for LC conditions that are outside of the plan are much less accurate and should be avoided. The final part of the work deals with the effect of extracolumn band dispersion on the peak capacity of linear gradients at various gradient times, mobile phase flow rates, and column temperature. The peak capacity determined for corrected experimental data were consistent with the published results dealing with the optimization of peak capacity in gradient elution.

Analysis of the solvent strength parameter (linear solvent strength model) for isocratic separations in reversed-phase liquid chromatography

The solvent strength parameter (slope) and intercept (log kw) of the linear solvent strength model are determined for methanol-water (17 columns), acetonitrile-water (15 columns), acetone-water (7 columns), tetrahydrofuran-water (6 columns), and 2-propanol-water (4 columns) for varied compounds. It is shown that the linear region of the plots of the retention factor (log k) against the volume fraction of organic solvent (ϕ) is largely a system property independent of solute type for compounds with accessible retention factors. Solvent strength parameters for methanol (3.12 ± 0.12), acetonitrile (2.78 ± 0.18), acetone (2.71 ± 0.11), and tetrahydrofuran (2.95 ± 0.24) are assigned for siloxane-bonded silica and porous graphitic carbon columns as an average of the compound-specific experimental values. Statistical analysis indicates that the compound-specific solvent strength parameters are column dependent and limited as a general solvent property. Evaluation of the column-specific solvent strength parameters provides further evidence that the average solvent strength parameter is column dependent. The column-specific solvent strength values are compared with the hypothetical water-organic solvent distribution systems using the system constants of the solvation parameter models as variables confirming the column dependence of the solvent strength parameter. The column-specific solvent strength parameters can be predicted by the solvation parameter model with a typical deviation of about 0.12 over a range of 1.69 to 6.33 for the experimentally determined values. The intercept of the linear solvent strength model is shown to be both column and organic solvent dependent. Compound column-specific values are adequately described by the solvation parameter model. Log kw cannot be recommended as a descriptor of solute properties since it has no clear connection to a real distribution system.

Prediction of n-octanol/water partition coefficient of strongly ionized compounds by ion-pair reversed-phase liquid chromatography with silica-based stationary phase

反相液相色谱(RPLC)是测定正辛醇/水分配系数(log P)的有效方法,但由于缺少同类型模型化合物,RPLC在测定强离解化合物的log P时遇到挑战。该文在硅胶基质C18色谱柱上,采用离子抑制反相液相色谱(IS-RPLC)和离子对反相液相色谱(IP-RPLC)分别对中性化合物、酚酸、羧酸、磺酸及部分两性化合物的保留行为进行了系统研究。在IS-RPLC模式下,利用中性化合物、弱离解的酚酸和苯羧酸作为模型化合物,建立了表观正辛醇/水分配系数(log D)与纯水相保留因子对数值(log kw)的定量结构-保留行为关系(QSRR)模型,测定了19种离解化合物的log D值,作为后续IP-RPLC的模型化合物及验证化合物。在IP-RPLC模式下,将中性、弱离解和强离解化合物作为混合模型组,以溶质静电荷ne、氢键酸碱性参数A和B为桥梁,建立了线性良好的log D-log kw-IP模型,采用3种不同类型的离解化合物进行了外部验证实验,预测值误差低于10%,证实了模型的可靠性。在此基础上,预测了8种强离解化合物的log D7.0值(pH 7.0条件下的log D值)。研究表明,利用结构相关参数沟通不同类型的模型化合物,是实现IP-RPLC测定强离解化合物log D值的一种行之有效的方法。与聚乙烯醇基质色谱柱相比,通用型的硅胶基质色谱柱上尽管存在着更多的次级作用,但可以为强离解化合物log D的测定提供更灵活的选择。

Effects of buffer salt types and non-counter ions of ion-pair reagents on the retention behavior of strongly ionized acid compounds in ion-pair reversed-phase liquid chromatography

在离子对反相液相色谱(IP-RPLC)分析中,溶质保留受对离子(counter ion)的影响比较受人关注,但鲜有研究流动相中缓冲盐类型和离子对试剂中非对离子(non-counter ion)对溶质保留行为的影响。鉴于此,该文以14种磺酸化合物为研究对象,甲醇为有机调节剂,分别考察了3种缓冲盐体系(磷酸二氢铵、氯化铵和乙酸铵)和5种离子对试剂体系(四丁基溴化铵、四丁基磷酸二氢铵、四丁基硫酸氢铵、四丁基硝酸铵和四丁基乙酸铵)下强离解酸性化合物的IP-RPLC保留行为,通过比较不同流动相条件下得到的溶质log kw(100%水相作流动相时的保留因子)、S(线型溶剂强度模型线性回归得到的常数),以及CHI(色谱疏水指数,log kw/S),寻找保留行为规律。研究表明,流动相中的缓冲盐类型和离子对试剂非对离子均会影响化合物的log kw和S值,所有化合物在氯化铵缓冲盐体系下具有最大的log kw值。相对于无机阴离子,离子对试剂中弱离解性有机阴离子(乙酸根)的存在有利于增加磺酸化合物的S值。通过对比不同条件下的保留行为,推测磺酸化合物的IP-RPLC保留机理中同时存在着离子对模型和动态离子交换模型。与log kw和S值不同,化合物的CHI值受缓冲盐类型以及离子对试剂非对离子的影响较弱。此外,研究发现化合物的表观正辛醇/水分配系数(log D)与log kw、S、CHI之间均具有良好的线性相关性。不同缓冲溶液和不同离子对试剂非对离子条件下获得的log kw和S值存在着一定的差异,而CHI值相对稳定,因此,CHI更适用于IP-RPLC中定量结构-保留行为关系模型的建立。

Closed form approximations to predict retention times and peak widths in gradient elution under conditions of sample volume overload and sample solvent mismatch

Closed form expressions for the prediction of retention times and peak widths for gradient liquid chromatography are particularly useful in understanding, rationalizing and optimizing separations. These expressions are obtained by integrating differential equations, in conjunction with a model of the variation of the retention factor as a function of mobile phase composition. Two of these models, the linear solvent strength (LSS) model and the Neue-Kuss (NK) model are explored in the present work. Here, we expand on these closed form expressions to account for effects of sample volume overload and a mismatch between the sample solvent and the initial mobile phase composition for the gradient. We show that there have been errors in expressions reported in the literature, and we have evaluated the accuracy of the predictions from the closed form expressions reported here using a recently developed liquid chromatography simulator. The expressions assume a constant plate height and consider elution across four zones of the gradient profile – elution in the sample solvent, elution in the initial (isocratic) mobile phase caused by the gradient delay volume, elution during a linear gradient, and elution post-gradient at the final (isocratic) mobile phase composition. The expressions generally give reasonably accurate predictions for retention times and peak widths, except for cases where the solute elutes during transitions between the different zones. The average magnitude of the prediction errors for retention time and peak width relative to simulation were 0.093% and 0.40% for the LSS expressions for ten amphetamine solutes at 36 different separation conditions, and 0.22% and 1.8% for the NK expressions for eight alkylbenzene solutes at 36 different separation conditions, respectively.

Numerical approximation of nonlinear and non-equilibrium model of gradient elution chromatography

A nonlinear lumped kinetic model of liquid chromatography is formulated and solved numerically to theoretically investigate the effect of column overloading on gradient elution. Linear solvent strength (LSS) model is utilized for Henry’s constant, non linearity coefficient and axial dispersion coefficient. A semi-discrete high-resolution finite volume scheme is extended and applied to obtain the approximate solutions of the governing model equations. The effects of changing modulator concentration are examined on the single and two-component elution. The benefits of gradient elution over isocratic elution are thoroughly discussed. The influences of optimizing free-parameters available in gradient chromatography are analyzed on the efficiency of the column and on the production of targeted components. For instance, the results obtained are used to study the effects of gradient slope, modulator concentration, solvent strength, nonlinearity coefficient, mass transfer coefficient, and axial dispersion coefficient on the concentration profiles.

Effects of peak compression in gradient elution of liquid chromatography

谱带压缩效应是梯度洗脱区别于等度洗脱的重要特征。经典的范德姆特(van Deemter)理论塔板高度方程基于等度洗脱推导得到,因此不能对谱带压缩效应进行描述。在梯度洗脱中,保留因子(k)会随流动相组成(φ)的改变而发生变化,这就使得对梯度洗脱机理的研究要比等度洗脱复杂许多。该文对近10年来谱带压缩效应的研究进展,特别是溶剂强度模型(即描述ln k与φ关系的数学表达式)的非线性特征对谱带压缩因子(G)的影响进行了述评,指出为了更好地认识谱带压缩效应需要将这种非线性因素考虑在内。

Testing experimental designs in liquid chromatography (I): Development and validation of a method for the comprehensive inspection of experimental designs

The basis of interpretive optimisation in liquid chromatography is the prediction of resolution, from appropriate solute retention models. The reliability of the process depends critically on the quality of the experimental design. This work develops, validates and applies a general methodology aimed to evaluate the quality of any training experimental design, which will be applied in Part II to design optimisation. The methodology is based on the systematic evaluation of the uncertainties associated to the prediction of retention times in comprehensive scans of both isocratic and gradient experimental conditions. It is able to evaluate comprehensively experimental designs of arbitrary complexity. Five common training experimental designs were used to model the retention, according to the Linear Solvent Strength (LSS) and the Neue-Kuss (NK) equations, using a set of 14 sulphonamides of different polarity. The results are presented in terms of relative uncertainties in predictions, which provide significant and interpretable results. The magnitude of such uncertainties, together with the systematic, coherent and logical changes observed at increasing solute hydrophobicity, give support to the results. The NK model gave smaller errors and unbiased predictions, whereas the LSS model gave rise to lack of fit. Isocratic training designs, which are widely accepted as the most informative, are confirmed as the best. As a general conclusion, gradients are predicted with intrinsically smaller uncertainties, independently of the training experimental design. In addition, gradients are more insensitive than isocratic predictions with regard to the type of training design used. Isocratic predictions deteriorate quickly with mobile phase composition. This explains the better performance of gradient predictions, even with biased models.

Determination of the Thermodynamic Behavior of a Therapeutic Peptide in Overloading Conditions in Gradient Elution Chromatography

The aim of this work is to model the adsorption behavior of a cyclic octapeptide, octreotide, in reversed-phase gradient elution liquid chromatography. Adsorption isotherms of the peptide were firstly determined in isocratic conditions, using different peptide concentrations and compositions of the mobile phase (mixtures with different percentage of organic modifier, ranging from 23 to 28% v/v). Inverse Method (IM) was applied to determine the parameters describing the isotherm equation for every composition of mobile phase considered. Then, the isotherm parameters have been correlated to the amount of organic modifier in the mobile phase, through the Linear Solvent Strength model (LSS). In the end, it was possible to predict the chromatographic behavior of the cyclic octapeptide in overloading gradient conditions, the knowledge of which can be useful when scaling the method in preparative conditions.

Influence of the pre-elution of solute in initial mobile phase on retention time and peak compression under linear gradient elution

Under gradient elution the solute will firstly migrate in initial mobile phase due to dwelling time (tD) of the system. The pre-elution of solute in initial mobile phase can be accounted for by a dimensionless value of tD/(t0kφ0), where t0 and kφ0 denote dead time and retention factor in initial mobile phase, respectively. The influence of tD/(t0kφ0) on retention time (tR) and peak compression factor (G) are discussed under linear gradient elution. A general expression for G is proposed, and two analytical forms of it which are suited for linear solvent strength model (LSSM) and quadratic solvent strength model (QSSM) respectively are presented. The well-known Poppe equation for G which is derived under LSSM is extended by taking tD/(t0kφ0) into account. The expression for G under QSSM takes into account the curvature in the plot of logarithmic retention factor vs. mobile phase composition. By taking twelve compounds degraded from lignin as analytes, the experimental and predicted chromatograms are found to be well consistent. It is shown that tD/(t0kφ0) has more effects on the prediction of tR than that of G.

Modeling the nonlinear behavior of a bioactive peptide in reversed-phase gradient elution chromatography

The thermodynamic behavior of octreotide, a cyclic octapeptide with important pharmaceutical functions, has been simulated under reversed-phase gradient elution conditions. To this end, adsorption behavior was firstly investigated in isocratic conditions, under a variety of water/acetonitrile + 0.02% (v/v) trifluoroacetic acid (TFA) mixtures as mobile phase by using a Langmuir isotherm. Organic modifier was varied in the range between 23 and 28% (v/v). Adsorption isotherms were determined by means of the so-called Inverse Method (IM) with a minimum amount of peptide. The linear solvent strength (LSS) model was used to find the correlation between isotherm parameters and mobile phase composition. This study contributes to enlarge our knowledge on the chromatographic behavior under nonlinear gradient conditions of peptides. In particular, it focuses on a cyclic octapeptide.

Extension of the linear solvent strength retention model including a parameter that describes the elution strength changes in liquid chromatography

Modelling the retention behaviour of solutes in liquid chromatography, based on the composition of the mobile phase is a common task in the chromatographic practice. Along the development of liquid chromatography (LC), several models have been proposed to help in understanding the retention mechanisms, and especially, allow the prediction of retention times with optimisation purposes. Particular models are used for different LC modes, such as normal phase (NPLC), reversed phase (RPLC), hydrophilic interaction (HILIC), and micellar (MLC). In this work, a general equation is proposed that includes a parameter (the elution degree, g), which characterises the way the elution strength varies with the modifier concentration. The elution degree adopts the value g = 1 when the system follows the linear solvent strength (LSS) model, where the elution strength is constant. When g > 1, the elution strength decreases, and for g < 1, it increases with the modifier concentration. The proposed equation was applied to experimental retention data obtained for several chromatographic systems in RPLC, HILIC, MLC, and microemulsion LC. It was found that values in the 1 < g < 2 range are most usual. The general behaviour of the proposed equation was studied for isocratic and gradient elution. A general expression to calculate the compression factor of chromatographic peaks in gradient elution was also developed. It is shown that an increasing g value makes retention factors close to zero more difficult, since the elution strength decreases as the modifier concentration increases. For this reason, the larger the g value, the harder it is to reach significant peak compression. In contrast, an elution mode with g < 1 would yield increased elution strength with the modifier concentration, giving rise to significant peak compression.

Peak dispersion in gradient elution: An insight based on the plate model

Gradient elution in liquid chromatography reduces the analysis time, improves the efficiency and increases the peak capacity. The study of this chromatographic mode has been based mainly on kinetic dispersion models. The plate model has been applied to a lesser extent, despite being the basis for the concepts of plate height and chromatographic efficiency. In this work, a general equation describing peak dispersion in HPLC gradient elution is derived from the plate model. This equation is studied and validated for three types of gradients: (i) a reference gradient without ramp in which the retention factor varies with time identically throughout the column, (ii) a gradient of stationary phase in which the nature of the stationary phase varies continuously inside the column, resulting in a ramp of constant retention factor over time, and finally, (iii) a mobile phase gradient, which produces a retention factor ramp that varies over time. In the latter case, the results are similar to those derived from the mass-transport equation in linear solvent gradients when the linear solvent strength model is applied. The final equations are expressed based on the initial and final instantaneous retention factors, thus they can be applied independently of the deviation of the elution model, being fully compatible with isocratic elution. Results predicted with the proposed equations are identical to those obtained by numerical resolution of the elution differential equation system. The additional compression due to the presence of a ramp of modifier is also verified. However, useful compressions will appear only when the retention factor changes with time. Finally, the study indicates that the extra-column variance undergoes a compression process when the retention factor varies over time, whether or not there is a ramp inside the column.

DynaStI: A Dynamic Retention Time Database for Steroidomics.

Steroidomics studies face the challenge of separating analytical compounds with very similar structures (i.e., isomers). Liquid chromatography (LC) is commonly used to this end, but the shared core structure of this family of compounds compromises effective separations among the numerous chemical analytes with comparable physico-chemical properties. Careful tuning of the mobile phase gradient and an appropriate choice of the stationary phase can be used to overcome this problem, in turn modifying the retention times in different ways for each compound. In the usual workflow, this approach is suboptimal for the annotation of features based on retention times since it requires characterizing a library of known compounds for every fine-tuned configuration. We introduce a software solution, DynaStI, that is capable of annotating liquid chromatography-mass spectrometry (LC–MS) features by dynamically generating the retention times from a database containing intrinsic properties of a library of metabolites. DynaStI uses the well-established linear solvent strength (LSS) model for reversed-phase LC. Given a list of LC–MS features and some characteristics of the LC setup, this software computes the corresponding retention times for the internal database and then annotates the features using the exact masses with predicted retention times at the working conditions. DynaStI is able to automatically calibrate its predictions to compensate for deviations in the input parameters. The database also includes identification and structural information for each annotation, such as IUPAC name, CAS number, SMILES string, metabolic pathways, and links to external metabolomic or lipidomic databases.

Gradient method transfer after changing the average pore diameter of the chromatographic stationary phase I – One-dimensional sample mixture

Three different approaches designed to transfer gradient methods from a chromatographic column 1 packed with particles (2.7 μm 90 ÅCORTECS-C18 or CORTECS-Triphenyl) to a column 2 packed with the same particles having a larger average pore diameter (APD = 120 Åand 450 Å) are proposed for a one-dimensional sample mixture. Two approaches are based on the variation of the experimental retention plots (lnk vs. the volume fraction, φ, of the strong solvent) with increasing the APD. They lead to so-called “vertical” (vertical shift in column phase ratio, lnϕ2ϕ1) and “horizontal” (horizontal shift in eluent composition, Δφ1 →2) gradient method transfers. The third method is based on in silico predictions of the gradient retention times when considering the actual non-linearity of the retention plots. The adjusted gradient parameters (starting eluent composition, φ0, and temporal gradient steepness, β) are unambiguously determined by minimizing the distance between the calculated and targeted gradient retention times of all the analytes.The performances of the three approaches for gradient method transfer are compared for a sample mixture containing a non-retained compound (thiourea) and a series of five homologous compounds (n-alkanophenones). The ultimate goal is to keep unchanged the gradient retention times of all analytes when changing the APD of the particles. The results show that the in silico transfer systematically outperforms the “horizontal” transfer, which itself outperforms the “vertical” transfer. The first two approaches are the least successful ones because, even for a series of homologous compounds, the linear solvent strength model (LSSM) is only an approximate model and the best shifts in eluent composition that keeps retention factors unchanged are compound-dependent. In the end, the average relative deviations between the observed and targeted gradient retention times are 15.0, 1.7, and 0.4% (90 Åto 120 Åtransfer, C18 chemistry), 5.1, 5.8 and 0.8% (90 Åto 450 Åtransfer, C18 chemistry), 4.8, 0.5, and 0.4% (90 Åto 120 Åtransfer, Triphenyl chemistry), and 10.4, 7.1, and 2.2% (90 Åto 450 Åtransfer, Triphenyl chemistry) for the “vertical”, “horizontal”, and “in silico” gradient method transfers, respectively.

A novel strategy for retention prediction of nucleic acids with their sequence information in ion-pair reversed phase liquid chromatography

In this work, retention behaviors of oligonucleotides and double-stranded deoxyribonucleic acids (dsDNAs) have been investigated in ion-pair reversed-phase liquid chromatography (IP-RPLC). We demonstrated that classic linear solvent strength (LSS) model is applicable for describing isocratic retention of oligonucleotides and dsDNAs, which indicated that nucleic acids share the similar retention mechanism as other common small molecules in IP-RPLC. The separation of nucleic acids in IP-RPLC is driven by both hydrophobic and electrostatic interactions. We defined the parameter lnkw/S obtained from LSS model in IP-RPLC as chromatographic hydrophobic and electrostatic interaction index (CHEI). CHEI of a nucleic acid has been revealed to correlate well with its gradient retention time. Notably, we proposed a strategy for retention prediction based on CHEI and base sequence information of nucleic acids. Corresponding to base locus, each base sequence is converted to a featured locus vector consisting of zeros and ones. CHEI prediction models were established by support vector regression (SVR) algorithm with locus vectors. Predicted CHEI values have been applied to predict retention times under desired gradient elution runs. This protocol is easy to grasp and worth pursuing further development for more precise retention prediction performance of nucleic acids in IP-RPLC.

Generic approach to the method development of intact protein separations using hydrophobic interaction chromatography.

We describe a liquid chromatography method development approach for the separation of intact proteins using hydrophobic interaction chromatography. First, protein retention was determined as function of the salt concentration by isocratic measurements and modeled using linear regression. The error between measured and predicted retention factors was studied while varying gradient time (between 15 and 120min) and gradient starting conditions, and ranged between 2 and 15%. To reduce the time needed to develop optimized gradient methods for hydrophobic interaction chromatography separations, retention-time estimations were also assessed based on two gradient scouting runs, resulting in significantly improved retention-time predictions (average error<2.5%) when varying gradient time. When starting the scouting gradient at lower salt concentrations (stronger eluent), retention time prediction became inaccurate in contrast to predictions based on isocratic runs. Application of three scouting runs and a nonlinear model, incorporating the effects of gradient duration and mobile-phase composition at the start of the gradient, provides accurate results (improved fitting compared to the linear solvent-strength model) with an average error of 1.0% and maximum deviation of -8.3%. Finally, gradient scouting runs and retention-time modeling have been applied for the optimization of a critical-pair protein isoform separation encountered in a biotechnological sample.

Applicability of retention modelling in hydrophilic-interaction liquid chromatography for algorithmic optimization programs with gradient-scanning techniques

Computer-aided method-development programs require accurate models to describe retention and to make predictions based on a limited number of scouting gradients. The performance of five different retention models for hydrophilic-interaction chromatography (HILIC) is assessed for a wide range of analytes. Gradient-elution equations are presented for each model, using Simpson’s Rule to approximate the integral in case no exact solution exists. For most compound classes the adsorption model, i.e. a linear relation between the logarithm of the retention factor and the logarithm of the composition, is found to provide the most robust performance. Prediction accuracies depended on analyte class, with peptide retention being predicted least accurately, and on the stationary phase, with better results for a diol column than for an amide column. The two-parameter adsorption model is also attractive, because it can be used with good results using only two scanning gradients. This model is recommended as the first-choice model for describing and predicting HILIC retention data, because of its accuracy and linearity. Other models (linear solvent-strength model, mixed-mode model) should only be considered after validating their applicability in specific cases.