Retention and column efficiency in reversed phase liquid chromatography as a function of pH for optimization purposes

Summary The effects of pH on both the solute retention and the peak shape of ionogenic compounds are studied in order to propose accurate models for pH optimization purposes. Several mathematical models (theoretical and empirical) for describing the variation of the retention factor versus pH are compared within different pH ranges. Limits of such models used for optimizing the pH by requiring only 3 preliminary experimental runs, are discussed in terms of deviations (≤±5%) of predicted retention times from experimental retention times. An original procedure is developed for selecting the most convenient retention model, from a given set of three retention data. This set is also applied to modeling the variation of both peak width and peak asymmetry with mobile phase pH conditions. Such a procedure is demonstrated as helpful for the separation of ionogenic solutes by considering mobile phase pH as an additional variable that can be useful during optimization procedures.     

反相高效液相三元流动相台阶梯度分离条件的快速优化方法

 根据溶质在柱内的迁移规律 ,建立了一种利用线性梯度实验快速获得溶质保留值方程系数 ,然后以串行响应函数为优化指标进行多台阶梯度分离条件优化的方法。与利用等度实验获得保留值方程的方法相比 ,该法可以大大缩短优化时间。通过该方法对芳香胺和衍生化氨基酸样品进行了分离 ,获得了满意的分离度 ,表明该方法的预测精度很好。     

可调移动重叠分离图法用于高效液相色谱多台阶梯度分离条件优化的研究

计算机辅助高效液相色谱（HPLC）分离条件优化可以低成本、快速地得到优化的分离条,因而已较为广泛地用于复杂样品的分离分析。基于移动重叠分离图方法,又发展了一种新型的多台阶梯度分离条件的优化方法可调移动重叠分离图法。该方法通过预测不同流动相条件下各组分的保留时间、峰宽和分离度,绘制出对于样品中各组分的重叠分离区域图。在对当前台阶流动相组成进行优化的同时,考虑其对后面一到两个台阶上流出组分保留的影响,实时地重新绘制对于后面台阶上流出组分的重叠分离区域图。通过观察当前台阶流动相条件对当前台阶和后面台阶上流出组分分离的影响,综合考虑样品中所有组分的分离情况,找到更接近全局最优的分离条件。通过扫描的方法对优化得到的分离条件进行微调,能够进一步提高分离效果。采用文献数据对可调移动重叠分离图法的应用加以说明,在二元流动相体系下,证明了该方法在HPLC方法建立方面的优越性。     

Ionisation Effects in the Reversed Phase Liquid Chromatographic Separation of Thyromimetic Iodoamino Acids

Abstract

The influence of ionisation equilibria on the retention behaviour of iodoamino acids and related compounds on micro-particulate octadecylsilica supports has been examined. The chromatographic data for these ionogenic solutes have been discussed in terms of current concepts for reversible solvophobic interactions with the hydrocarbonaceous stationary phase. This treatment permits the conditional effects of the mobile phase composition and pH on solute retention to be assessed and the relationship between the molecular surface area of a solute and its retention to a non polar stationary phase evaluated.     

Contributions to reversed-phase column selectivity. I. Steric interaction

In reversed-phase chromatography (RPC), the restricted retention of "bulky" solutes can occur in one of two ways, giving rise to either "shape selectivity" or "steric interaction." Starting with data for 150 solutes and 167 monomeric type-B alkylsilica columns, the present study examines the steric interaction process further and compares it with shape selectivity. The dependence of column hydrophobicity and steric interaction on column properties (ligand length and concentration, pore diameter, end-capping) was determined and compared. The role of the solute in steric interaction was found to be primarily a function of solute molecular length, with longer solutes giving increased steric interaction. We find that there are several distinct differences in the way shape selectivity and steric interaction are affected by separation conditions and the nature of the sample. Of particular interest, steric interaction exhibits a maximum effect for monomeric C(18) columns, and becomes less important for either a C(1) or C(30) column; shape selectivity appears unimportant for monomeric C(1)-C(18) columns at ambient and higher temperatures, but becomes pronounced for C(30) - as well as polymeric columns with ligands ≥C(8). One hypothesis is that shape selectivity involves the presence or creation of cavities within the stationary phase that can accommodate a retained solute (a primarily enthalpic process), while steric interaction mainly makes greater use of spaces that pre-exist the retention of the solute (a primarily entropic process). The related dependence of hydrophobic interaction on column properties was also examined.     

The determination of starting conditions for modifier optimization in reversed-phase HPLC

Summary After a brief introduction of the subject, the paper focusses on the first step in any optimization procedure: the delineation of the parameter space, wherein the global optimum is to be found. For organic modifier optimization in reversed-phase liquid chromatography it is shown that the necessary information can be derived from a single water-methanol gradient. It first yields an estimate of the total number of solutes in the sample, which is vital to define the peak capacity needed to achieve separation at a certain confidence level. Next, the gradient allows the prediction of suitable isocratic methanol binary solvents, and transfer rules formulate the iso-eluotropic composition of the common binary solvents (tetrahydrofuran and acetonitrile). Because all predictions are based on a statistical analysis of a limited data base, attention is given to the practical situation where an actual sample deviates from the average solute behaviour. Such deviations are revealed in the first isocratic run and can be used to arrive at a better estimate of solute retention.     

Reversed-Phase Liquid Chromatography with Mixed Eluents: Partition Model of Retention for Ionogenic Solutes

Abstract

Basing on the pure partition model of solute retention new theoretical equations for ionogenic solutes are discussed. These equations define dependence of the capacity ratio on the mobile phase composition, which is characteristic for the reversed-phase liquid chromatography.     

Analysis of selected ionic liquid cations by ion exchange chromatography and reversed-phase high performance liquid chromatography

The chromatographic behavior of 8 ionic liquids - 7 homologues of 1-alkyl-3-methylimidazolium and 4-methyl-N-butylpyridinium - has been investigated with a strong cation exchange adsorbent. In particular, the dependence of the retention properties of these solutes on mobile phase composition, pH, and buffer concentration was evaluated with the aim of optimizing and improving the selectivity and retention of solute separation. While using the SCX stationary phase, several interactions occurred with varying strengths, depending on the mobile phase composition. Cation exchange, nonspecific hydrophobic interactions, and adsorption chromatography behavior were observed. Reversed phase chromatography occurred at low concentrations of acetonitrile, electrostatic and adsorption interactions at higher organic modifier concentrations. Elevated buffer concentrations lowered the retention factors without affecting the selectivity of ionic liquids. Obtained results were further compared to the chromatographic behaviour of ionic liquids in the reversed phase system. All analyzed ionic liquids follow reversed-phase behavior while being separated. Much lower selectivity in the range of highly hydrophilic compounds is obtained. This suggests preferred use of ion chromatography for separation and analysis of compounds below 4 carbon atoms in the alkyl side chain.     

Retention modeling and simultaneous optimization of pH value and gradient steepness in RP‐HPLC using feed‐forward neural networks

A novel approach is proposed for the simultaneous optimization of mobile phase pH and gradient steepness in RP‐HPLC using artificial neural networks. By presetting the initial and final concentration of the organic solvent, a limited number of experiments with different gradient time and pH value of mobile phase are arranged in the two‐dimensional space of mobile phase parameters. The retention behavior of each solute is modeled using an individual artificial neural network. An “early stopping” strategy is adopted to ensure the predicting capability of neural networks. The trained neural networks can be used to predict the retention time of solutes under arbitrary mobile phase conditions in the optimization region. Finally, the optimal separation conditions can be found according to a global resolution function. The effectiveness of this method is validated by optimization of separation conditions for amino acids derivatised by a new fluorescent reagent.     

Column wall modifications using polyethylene oxide for capillary electrophoresis of ribonucleotides

Summary In this study, a variety of fused silica capillaries with different combinations and sequences of treatments with HMDS and polyethylene oxide were prepared in order to develop an optimized column modification method for analysis of ribonucleotides. The 12 most common ribonucleotides (UTP, CTP, ATP, GTP, UDP, CDP, ADP, GDP, UMP, CMP, AMP, and GMP) in human cells were used as test solutes. Column performance measurements, including electroosmotic flow (EOF), solute migration speed and retention, column efficiency, peak shape, and resolution were investigated. By analyzing solute migration speed and retention of various hydrophilic/hydrophobic solutes, the column wall effects (EOF and adsorption) can be distinguished. This analysis method can give guidance in optimizing polymer coating properties (hydrophilicity/hydrophobicity) for CE columns. By studying the performance of these columns after various surface treatments, we were able to improve the separation of ribonucleotides from real samples to within 16 minutes with high efficiency and stability (over 300 analyses) using columns first deactivated with hexamethyldisilazane, and then coated with polyethylene oxide.     

Retention behaviour of imidazolium ionic liquid cations on 1.7 μm ethylene bridged hybrid silica column using acetonitrile-rich and water-rich mobile phases

The potential of 1.7 μm ethylene bridged hybrid silica phase was investigated for the separation of twelve imidazolium-based ionic liquid cations. U-shaped retention profile was observed for all solutes with an increase in retention at both low and high acetonitrile content. Chromatographic behaviour of imidazolium cations in both hydrophilic interaction chromatography (HILIC) and per aqueous liquid chromatography (PALC) modes was studied by varying key parameters such as buffer concentration and pH, acid additive, organic modifier and column temperature. Experimental data provided some evidences that under PALC conditions cationic solutes are retained predominantly by mixed hydrophobic/ion-exchange interactions. In the HILIC mode, both partitioning and ion-exchange interactions are responsible for the retention of solutes. Compared to PALC, HILIC provided significantly higher efficiencies with less or even no peak tailing, better separation selectivity and greater resistance to overload. In PALC mode gradient elution was required to achieve adequate retentivity of all solutes but selectivity was not sufficient to distinguish between solutes with very similar hydrophobicity. In contrast, under HILIC conditions twelve solutes were almost completely resolved in less than 4 min by using isocratic elution. Summarizing, it could be concluded that ethylene bridged hybrid silica column providing a dual retention mechanism offers the possibility of selecting between the two retention modes with opposite separation selectivity, just by changing the composition of the mobile phase.     

Is hydrophilic interaction chromatography with silica columns a viable alternative to reversed-phase liquid chromatography for the analysis of ionisable compounds?

The separation of acidic, neutral and particularly basic solutes was investigated using a bare silica column, mostly under hydrophilic interaction chromatography (HILIC) conditions with water concentrations >2.5% and with >70% acetonitrile (ACN). Profound changes in selectivity could be obtained by judicious selection of the buffer and its pH. Acidic solutes had low retention or showed exclusion in ammonium formate buffers, but were strongly retained when using trifluoroacetic acid (TFA) buffers, possibly due to suppression of repulsion of the solute anions from ionised silanol groups at the low (s)(s)pH of TFA solutions of aqueous ACN. At high buffer pH, the ionisation of weak bases was suppressed, reducing ionic (and possibly hydrophilic retention) leading to further opportunities for manipulation of selectivity. Peak shapes of basic solutes were excellent in ammonium formate buffers, and overloading effects, which are a major problem for charged bases in RPLC, were relatively insignificant in analytical separations using this buffer. HILIC separations were ideal for fast analysis of ionised bases, due to the low viscosity of mobile phases with high ACN content, and the favourable Van Deemter curves which resulted from higher solute diffusivities.     

The role of mobile-phase pH in the determination of catecholamine-related compounds by reversed-phase liquid chromatography with amperometric detection

Summary A study was conducted for the optimization of the reversed-phase high performance liquid chromatographic separation of different types (acidic, basic, amphoteric and neutral) of catechol-related compounds by varying the pH. For pH to be used as the optimizing parameter, its effects on retention, peak width and peak shape had to be investigated. Therefore, the pH dependence of the amperometric detector response, as measured by the oxidation peak height or peak area, was taken into account.     

Contributions to reversed-phase column selectivity. II. Cation exchange

The contribution of cation exchange to solute retention for type-B alkylsilica columns (made from high-purity silica) has been examined in terms of the hydrophobic-subtraction (H-S) model of reversed-phase column selectivity. The relative importance of cation exchange in the separation of ionized bases by reversed-phase chromatography (RPC) varies with (a) column acidity (values of the column cation-exchange capacity C), (b) mobile-phase pH and buffer concentration, and (c) the nature of the buffer cation. The effects of each of these separation variables on cation retention were examined. The contribution of cation exchange (and other ionic interactions) to solute retention is represented in the H-S model by properties of the solute (κ') and column (C), respectively. Values of κ' for 87 solutes have been examined as a function of solute molecular structure, and values of C for 167 type-B alkylsilica columns have been related to various column properties: ligand length (e.g., C(8) vs. C(18)) and concentration (μmol/m(2)), pore diameter (nm), and end-capping. These results contribute to a more detailed picture of the retention of cationic solutes in RPC as a function of separation conditions. While previous work suggests that the ionization of type-B alkylsilica columns is generally negligible with mobile-phase pH<7 (as a result of which cation exchange then becomes insignificant), the present study provides evidence for cation exchange (and presumably silanol ionization) at a pH as low as 3 for most columns.     

Optimization of a multisolvent composition in RPHPLC: Generalization to non- ideal peaks

Summary When samples contain solutes at very different concentrations, the use of the classical resolution function to express the quality of the separation does not provide a true measure of peak overlap and is therefore unsuitable for optimization. Apart from this case, the validity of the optimum is highly dependent on the accuracy of the predicted retention surfaces and it is essential to restrict the parameter space in which optimum performance is sought. As a result, most interpretive optimization procedures become unreliable for optimizing the composition of multisolvent systems in RPHPLC, particularly for non-ideal peaks. These difficulties can be overcome by the method proposed here: the ability, by means of a criterion, called CRIT, to select a restricted range of composition in order to enhance the accuracy of the response surface is developed for non-ideal peaks. The best composition within this selected range is then determined by using a modified resolution function. The performance of this procedure is discussed with some illustrative examples.     

Separation of basic solutes by reversed-phase capillary electrochromatography

The separation of basic solutes at low pH by capillary electrochromatography (CEC) has been investigated. The feasibility of separation of basic solutes by CEC was demonstrated. Influence of operational parameters, solvent composition, pH, temperature on retention and selectivity of the separation of a mixture of basic, neutral and acidic drug standards has been investigated. The observed elution behavior has been modeled to account for both chromatographic retention and differential electrophoretic mobility of the solutes. This model was verified experimentally. It is demonstrated in this work that the elution window of solutes in reversed-phase CEC is expanded to range from -1 to infinity.     

Effects of pH in reversed-phase liquid chromatography

The effects of concomitant variations in pH and organic modifier concentration on retention, efficiency and peak symmetry are considered for reversed-phase liquid chromatography (RPLC) on octadecyl-modified silica (ODS) columns. A number of factors are discussed, which make the systematic exploitation of pH effects in RPLC more complicated than the optimization of solvent composition. If the pH is varied, a second factor (usually the concentration of organic modifier) will need to be varied simultaneously to maintain retention (capacity factors) in the optimum range. When pH is considered as a parameter in RPLC, not only its effects on retention, but also the variations in efficiency (plate count) and peak shape (asymmetry) need to be considered. These parameters turn out to vary drastically between individual solutes and between different experimental conditions. The results of a study involving a number of acidic, basic and neutral solutes, two different ODS columns and mixtures of either methanol or acetonitrile with aqueous buffers are reported. In the earlier part of the study, using methanol as the organic modifier, reproducible data for retention, peak width and peak symmetry were obtained and these data are reported. In the later part of the study, using acetonitrile, a gradual change in retention as a function of time was observed, this effect coinciding with a decrease in column efficiency. It is concluded that ODS columns are subject to considerable degradation during studies in which the pH is varied. Although this effect can be described mathematically, the preferred solution is thought to be the use of pH-stable columns.     

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, pKa, etc.) on 10 different C18 columns (other conditions constant). These data have been combined with k values from a previous study (86 solutes, five different C8 and C18 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.     

Retention behavior of large polycyclic aromatic hydrocarbons on metalloprotoporphyrin-silica stationary phases

The retention behavior of large polycyclic aromatic hydrocarbons (LPAHs) (> or = 7 rings) on newly developed metalloplotoporphyrin (MProP)-silica stationary phases is examined and the results are compared to previously reported data for retention of the same solutes on commercially available phases. HPLC columns packed with FeProP-silica are shown to exhibit unique shape selectivity for LPAH retention, with the planar LPAHs always retained much longer than corresponding non-planar solutes. Solute planarity, length to breadth ratio (L/B value), and number of carbon atoms within the LPAHs are all demonstrated to contribute to the retention sequence observed. Further, the retention of LPAH solutes on FeProP-silica phases is shown to be more predictable than on other reversed-phase columns, with the elution sequence constant regardless of the mobile phase composition. Due to the extremely high planar selectivity of FeProP-silicas with respect to LPAH retention, it is envisioned that columns packed with these phases could be used in conjunction with existing commercial columns to devise Inethods for more efficient separation of complex mixtures of LPAHs in environmental and other samples.