Prediction of internal standards in reversed-phase liquid chromatography: IV. Correlation and prediction of retention in reversed-phase ion-pair chromatography based on linear solvation energy relationships

This paper describes the results of the evaluation of a new solvation parameter model for reversed-phase ion-pair chromatography by linear gradient elution. This model is described as . The first six terms are the usual solvation parameter equation for neutral solutes, and the seventh term represents the contribution to retention from solute’s ionization. The last term describes the retention increase due to ion-pair effect. Retention times obtained for 60 solutes (neutral, acidic and basic) in acetonitrile/aqueous mobile phases with different ion-pair reagents (phosphoric acid, trifluoroacetic acid, heptafluorobutyric acid, perchloric acid, and hexafluorophosphoric acid) are used to evaluate the capability of the function. It is concluded that the model describes the retention of ionizable/ionized compounds under ion-pair conditions very well. Accordingly, the function extends the application of linear solvation energy relationships (LSERs) to ionizable compounds in ion-pair chromatography, and allows us to easily predict their retention for chromatographic optimization, including selectivity optimization and internal standard selection. Finally, the conclusion can be extended to ioscratic elution.     

Semi-Preparative Isolation of Crocins from Saffron (Crocus sativus L.)

Two-step, low-pressure liquid chromatography has been used to isolate crocins, the most abundant compounds in the hydroalcoholic extract of saffron. A mixture of styrene–divinylbenzene copolymer and modified starch polymers was used as stationary phase. A 600 mm × 15 mm i.d. column was used in the first step; the best resolution was achieved by gradient elution with an aqueous methanol gradient with the methanol content changing from 50 to 95%. The highest yield of crocins from this step was achieved by use of 20 mL of 300 mg mL^?1 saffron extract. Under these conditions the yellow pigments of the saffron extract were separated into twenty fractions. In the second step use of a 700 mm × 8 mm i.d. column resulted in crocin-1 [crocetin (di- -D-gentobisyl) ester], crocin-2 [crocetin (mono- -D-gentobisyl-mono- -D-glycoside) ester], and crocin-3 [crocetin ( -D-gentobisyl)-ester] with purity as high as 99.04%, 97.40%, and 96.70%, respectively.     

Search for plant-induced technetium species with size-exclusion and reversed-phase ion-pair chromatography

In this work an attempt is made to have a closer look at technetium species (TcX) induced by spinach plants, grown on TcO _? ⁴ containing nutrient solutions. To this end, size-exclusion chromatography (SEC) and reversed-phase ion-pair chromatography (RP-IPC) were used: The performance was established with model compounds, i.e., anionic Tc-DTPA and cationic Tc-cyclam, used as mimics for TcX. Under the conditions applied, the information retrieved from high performance RP-IPC was minimal due to strong interactions of the technetium species with the Nucleosil C18 stationary phase. However, in low-pressure SEC non-size effects allowed differentiation of TcX into two distinct classes of technetium compounds, i.e., TcX₁ and TcX₂. From retention behavior in SEC, it was possible to speculate on the chemical properties of these two technetium species.     

Validated LC Method for Determination of Enantiomeric Purity of Apremilast Using Polysaccharide-Type Stationary Phases in Polar Organic Mode

Enantioseparation of an anti-psoriatic agent, apremilast (APR), was performed by HPLC using polysaccharide-type chiral stationary phases in polar organic mode for the first time. The separation capability of six different chiral columns (Chiralpak AD, Chiralpak IA, Chiralpak AS, Lux Amylose-2, Chiralcel OD and Chiralcel OJ-H) was investigated using neat MeOH and ACN. During the preliminary experiments the best results were obtained on Chiralpak IA column with ACN (R_s?=?5.4). The effects of binary mobile phases on the resolutions and retention factors were also investigated containing different percentages of MeOH:ACN. U-shaped retention pattern was obtained when plotting the retention factors of the APR enantiomers versus the MeOH content of the binary mobile phases on Chiralpak IA column. For further method optimizations an L25 orthogonal array table was employed altering the concentration of MeOH in ACN, column temperature, and flow rate. The best result was achieved on Chiralpak IA column with 80/20 (v/v%) MeOH/ACN with 0.7 mL min^?1 flow rate at 25 °C (R_s?=?5.4, t₂?=?7.45 min). Thermodynamic analysis revealed an enthalpy-driven enantioseparation. The developed HPLC method was validated according to the ICH guideline Q2(R1) and proved to be reliable, linear, precise and accurate for the determination of 0.1% R-enantiomer as chiral impurity in S-APR as well as quantification of the S-enantiomer.

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Prediction of internal standards in reversed-phase liquid chromatography. III. Evaluation of an alternative solvation parameter model to correlate and predict the retention of ionizable compounds

This paper describes the results of the evaluation of an alternative solvation parameter model for ionizable compounds. The new model is described as Log(k) = Int + rR2 + spi2(H) + asigmaalpha2(H) + bsigmabeta2(H) + mVx + U/(1 + V10 (+/-(pH-Pk))). The first six terms are the usual solvation parameter equation for neutral solutes, and the last term represents the contribution to retention from the ionization of solutes. Retention data obtained for 30 solutes in acetonitrile/aqueous buffer mobile phases are used to evaluate the capability of the function using different pH/pK scales. Because the function is not linear, nonlinear least-squares analysis is used to perform the data processing. It is concluded that the model function describes similarly the retention of ionizable compounds to the literature model without the need to accurately measure the mobile phase pH and solute's pK. Accordingly, the function simplifies the application of linear solvation energy relationships (LSERs) to ionizable compounds, and allows us to easily predict their retention for chromatographic optimization.     

Combined effect of organic modifier concentration and column temperature on retention in reversed-phase ion-pair liquid chromatography

Summary The retention behavior of phenylamine and naphthylamine sulphonic acid was evaluated in reversed-phase ion-pair liquid chromatography as a function of organic modifier concentration and column temperature. It has been observed that the logarithm of capacity factors decrease linearly with organic modifier concentration, and there is a good linear relationship between the intercept and slope for this relationship. Phenylamine and naphthylamine sulphonic acid retention decreases with increase in column temperature. A linear dependence of lnk _ip on the reciprocal of the absolute temperature, the Van't Hoff plot, was observed over the column temperature range studied, and the standard enhalpic change (H^o) for these sulphonic acid transfers from the mobile phaser to the stationary phase was determined. H^o was dependent on the solute structure and in the range from 2.5 Kcal/mol to 5.5 Kcal/mol, which is close to that observed in RP-HPLC. The enthalpy/entropy compensation effect was evaluated by plotting lnk _ip(T) vs. –H^o, and the apparent differences in retention mechanisms between the analytes were observed, which may arise from the significant differences in their configuration, hydrophobicity and the charges of the solutes as well as the complex retention processes of RP-IPC.     

反相离子对色谱法分析3′, 5′反向寡核苷酸

3′,5′反向寡核苷酸是碱基组成和长度完全相同、碱基顺序相反的两个寡核苷酸序列。以三乙胺为离子对试剂,研究了缓冲液浓度(0.025～0.15 mol/L)、pH (5.0～6.8)、柱温(25～45 ℃)、流速(0.3～0.7 mL/min)以及不同初始洗脱强度和洗脱梯度条件下,6个3′,5′反向寡核苷酸模拟样品保留和分离的变化特点。三组反向序列在缓冲液浓度为0.05 mol/L,pH 6.8和流速0.4 mL/min条件下获得最大分离,温度对分离的影响不大,而初始洗脱强度对反向序列的影响远大于洗脱梯度。实验结果表明3′,5′反向寡核苷酸的分离和保留趋势不完全一致,色谱条件的优化应有利于实现样品在柱上的弱保留。研究结果还显示寡核苷酸序列中5′末端的保留强于3′末端。     

Retention of ionizable compounds on HPLC. Modelling retention for neutral and ionizable compounds by linear solvation energy relationships

Summary A global LSER model that relates HPLC retention to mobile phase composition and pH is tested for a varied group of solutes, both neutral and ionizable, in a polymeric column and methanol-water mobile phases. It is compared to the local LSER model developed only for a given mobile phase, i.e., a fixed organic modifier content, and to the global LSER model set only for neutral solutes. The global LSER model for neutral and ionizable solutes requires a few supplementary parameters over the other models tested, but it accounts for retention under any experimental conditions for a given column and methanol-water mobile phases, describing properly the interactions established in the HPLC system (hydrophobicity, hydrogen-bond acidity and basicity, dipolarity/polarizability…). This paper is number 13 of a series with the same general title: “Retention of Ionizable Compounds on HPLC” published in various journals.     

反相离子对色谱法分析3′, 5′反向寡核苷酸

3′,5′反向寡核苷酸是碱基组成和长度完全相同、碱基顺序相反的两个寡核苷酸序列。以三乙胺为离子对试剂,研究了缓冲液浓度(0.025～0.15 mol/L)、pH (5.0～6.8)、柱温(25～45 ℃)、流速(0.3～0.7 mL/min)以及不同初始洗脱强度和洗脱梯度条件下,6个3′,5′反向寡核苷酸模拟样品保留和分离的变化特点。三组反向序列在缓冲液浓度为0.05 mol/L,pH 6.8和流速0.4 mL/min条件下获得最大分离,温度对分离的影响不大,而初始洗脱强度对反向序列的影响远大于洗脱梯度。实验结果表明3′,5′反向寡核苷酸的分离和保留趋势不完全一致,色谱条件的优化应有利于实现样品在柱上的弱保留。研究结果还显示寡核苷酸序列中5′末端的保留强于3′末端。     

Determination of t,t-muconic acid in urine samples using a molecular imprinted polymer combined with simultaneous ethyl chloroformate derivatization and pre-concentration by dispersive liquid–liquid microextraction

The present communication describes the preparation and evaluation of a molecularly imprinted polymer (MIP) as a solid-phase extraction (SPE) sorbent and simultaneous ethyl chloroformate (ECF) derivatization and pre-concentration by dispersive liquid–liquid microextraction (DLLME) for the analysis of t,t-muconic acid (t,t-MA) in urine samples using gas chromatography–mass spectrometry. The imprinting polymer was prepared using methacrylic acid as a functional monomer, ethylene glycol dimethacrylate as a cross-linker, 2,2-azobisisobutyronitrile as the initiator and t,t-MA as a template molecule. The imprinted polymer was evaluated for its use as a SPE sorbent by comparing both imprinted and non-imprinted polymers in terms of the recovery of t,t-MA from urine samples. Molecular modelling studies were performed in order to estimate the binding energy and efficiency of the MIP complex formed between the monomer and the t,t-MA. Various factors that can affect the extraction efficiency of MIP, such as the loading, washing and eluting conditions, were optimized; other factors that can affect the derivatization and DLLME pre-concentration were also optimized. MIP in combination with ECF derivatization and DLLME pre-concentration for t,t-MA exhibits good linearity, ranging from 0.125 to 2 μg?mL^?1 (R ²?=?0.9971), with limit of detection of 0.037 μg?mL^?1 and limit of quantification of 0.109 μg?mL^?1. Intra- and inter-day precision was found to be <6 %. The proposed method has been proven to be effective and sensitive for the selective pre-concentration and determination of t,t-MA in urine samples of cigarette smokers.

Thermodynamic transfer functions for urea and thiourea from water to water-tetrahydrofuran mixtures from precise vapor-pressure measurements

The rate of change of the standard chemical potential with solvent composition, \(\partial \bar G_0 /\partial Z\) , has been calculated from precise vapor-pressure measurements for urea at three temperatures and for thiourea at 298.15°K in water-tetrahydrofuran (THF) mixtures. From these results the standard free energy of transfer ΔG _t ^o of the solutes from water to various water-THF mixtures has been obtained together with the standard molar entropy ΔS _t ^o and the standard molar enthalpy ΔH _t ^o of transfer at 298.15°K in the case of urea. The quantity ΔG _t ^o for urea is negative in the water-rich region and positive for mole fractions THF>0.2. There is a nearly complete compensation between ΔH _t ^o andTΔS _t ^o at 298.15°K up to mole fraction THF=0.5. These phenomena can be partly related to the structure in H₂O-THF mixtures.     

The effects of pH and alcoholic organic modifiers on the direct separation of some acidic,basic and neutral compounds on a commercially available ovomucoid column

Summary The effects of alcoholic modifiers and pH on the chromatographic properties of an immobilized ovomucoid chiral stationary phase have been investigated using acidic, basic and neutral solutes. A series of primary, secondary and tertiary alcohols and pH's ranging from 3.5 to 6.0 were used in this study. The results indicate that both the shape and the hydrophobicity of the alcoholic modifier affect retention (k') and enantioselectivity (). In general, an increase in the hydrophobicity of the modifier results in a decrease in k's and 's. However, this is not the case whent-butanol is the modifier, suggesting that the size of the alkyl moiety attached to the carbinol carbon also contributes to the chromatographic results. The pH studies indicated that Coulombic interactions play a role in the retention of the acidic and basic solutes. The results also suggest that in addition to ethanol and 1-propanol,t-butanol should be considered during optimization and that maximum efficiencies may be obtained at pH 5.0.     

Spectrophotometric determination of glimepiride in pharmaceutical preparations based on the formation of charge-transfer and ion-pair complexes

Two rapid, simple, accurate and sensitive spectrophotometric methods were developed for the determination of glimepiride in pharmaceutical preparations. The first method was based on the formation of a charge-transfer complex of the drug, as n-electron donor, with 7,7,8,8-tetracyanoquinodimethane (TCNQ), as π-acceptor. The second method was based on the formation of ion-pair complexes between the examined drug and bromothymol blue (BTB). The proposed methods were validated for linearity, limit of detection, limit of quantification, precision, accuracy, robustness and specificity. The calibration was linear over the concentration range of 10–80 and 20–120 μg/mL for methods I and II, respectively. The limits of detection were 2.6 and 2.8 μg/mL. The proposed methods were applied to the determination of the drug in pharmaceutical preparations. The results obtained were in good agreement with those obtained using the reference method (HPLC). There was no significant difference in the accuracy and precision as revealed by the accepted values of t- and F-tests, respectively.     

One-Step Solvothermal Synthesis of Sub-2-µm Sea Urchin-Like TiO2 Microspheres for High-Performance Liquid Chromatography Stationary Phase

Sub-2-µm sea-urchin-like TiO₂ microspheres were prepared through the one-step solvothermal method. Results of scanning electron microscopy (SEM) and transmission electron microscopy (TEM) show that there are a lot of nanorods coming out from the center of the TiO₂ microsphere, making it look like a sea urchin. The morphology and structure of these microspheres are very suitable for chromatographic packing. During the separation of inorganic anions on this new stationary phase, the retention mechanism is electrostatic interaction, and the pH of the mobile phase plays an important role in retention behaviors. With this new stationary phase for separation of organic anions and nucleobases, Lewis acid–base interaction is more outstanding, which can cause peak broadening and tail for larger conjugated structure compounds. The separation of aromatic hydrocarbons on this new stationary phase demonstrates that Lewis acid–base interaction has a favorable influence on the separation of electron-rich aromatic hydrocarbons under normal phase chromatographic conditions.

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反相离子对色谱中有机溶剂浓度和离子对试剂浓度对保留值的影响

我们测定了氨基苯磺酸和氨基萘磺酸在反相离子对色谱中不同乙腈/水, 甲醇/水配比和离子对试剂浓度下的保留值,并把两种冲洗剂下的保留值和关系式lnk'=a+C~c~b中的参数a,C作线性关联,得到很好的相关性,这表明有机溶剂乙腈和甲醇对选择性并无显著的影响,但乙腈有更大的冲洗强度.证明关系式lnk'=A+Blnc~p+C~c~ b能较好地描述有机溶剂和离子对试剂浓度对保留值的影响, 但当离子对试剂浓度较高时该关系式不成立.同时提出了有机溶剂浓度和离子对试剂浓度"等同效应" 的概念     

Development of a Liquid–Liquid Microextraction Method Based on a Switchable Hydrophilicity Solvent for the Simultaneous Determination of 11 Drugs in Urine by GC–MS

A new and green liquid–liquid microextraction method based on a switchable hydrophilicity solvent for the determination of 11 drugs in urine is presented for the first time. The protonated N,N-dimethylcyclohexylamine was used as an extraction solvent and sodium hydroxide was injected as a trigger for organic-phase separation. The extraction procedure has been optimized and investigated in this study. The intraday and interday recovery ranged from 73.8 to 103.0% and the relative standard deviations were in the range from 2.2 to 13.5% (n?=?5). The linear range was from 5.0 to 2000.0 µg L^?1, and R² was greater than 0.99, and the limit of detection was in the range from 0.36 to 12.50 µg L^?1. The proposed method may be a useful procedure for the determination of 11 drugs in urine, and easily incorporated into routine testing for a laboratory.

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