Simultaneous separation of nonionic surfactants and polyethylene glycols by reversed phase high performance liquid chromatography

Summary The simultaneous separation of polyethylene glycol and its derivatives such as the lauryl alcohol and lauric acid ethoxylate oligomers was carried out by reversed phase high performance liquid chromatography. Branched fluorinated silica gel columns combined with evaporative light scattering detection were used for the characterization of nonionic surfactants. Lauryl alcohol ethoxylate oligomers were separated at 10°C with an isocratic eluent according to ethoxylate number and the retention time of the oligomers decreases with increasing ethoxylate number. The Van’t Hoff plots of retention factor of lauryl alcohol ethoxylate gave a complex cure, which is anomalous behavior for reversed phase high performance liquid chromatography. The anomalous Van’t Hoff plots were explained by a partial conformational change from polar to less polar conformers with increasing temperature. The most significant features for the analysis of the lauryl alcohol ethoxylate were the use of acetonitrile as mobile phase and operating temperature. The polyethylene glycol was separated according to ethoxylate number and the retention time of oligomers increased with increasing ethoxylate number. The Van’t Hoff plots of retention factor of polyethylene glycol had negative slopes. It was presumed that the polar conformation of the ethylene oxide chain decreased with increasing temperature. The lauryl alcohol ethoxylate and polyethylene glycol were separated simultaneously in gradient elution as a result of the conformational change of the ethylene oxide chain. As a practical example, lauric acid ethoxylate simultaneously separated into free polyethylene glycol, ethoxylate monolaurate and ethoxylate dilaurate in gradient elution.     

Comparison of reversed‐phase liquid chromatography and hydrophilic interaction chromatography for the fingerprint analysis of Radix isatidis

Radix isatidis is a famous anti‐influenza virus herbal medicine traditionally taken as a water decoction. However, the chemical fingerprint analysis of Radix isatidis is dominantly based on RPLC, from which it is difficult to obtain fingerprint information of hydrophilic compounds. Here, we developed the separation of Radix isatidis by RPLC and hydrophilic interaction chromatography, comparing the traditional RPLC fingerprint with the hydrophilic interaction chromatography fingerprint. Besides, an anti‐viral assay of Radix isatidis was conducted to evaluate its efficacy. The fingerprint–efficacy relationships between the fingerprints and the anti‐viral activity were further investigated with principal component regression analysis. The results showed that the anti‐viral activity correlated better with the hydrophilic interaction chromatography fingerprint than with the RPLC fingerprint. This study indicates that hydrophilic interaction chromatography could not only be a complementary method to increase the fingerprint coverage of conventional RPLC fingerprint, but also can better represent the efficacy and quality of Radix isatidis.     

Imidazolium‐embedded iodoacetamide‐functionalized silica‐based stationary phase for hydrophilic interaction/reversed‐phase mixed‐mode chromatography

A novel imidazolium‐embedded iodoacetamide‐functionalized silica‐based stationary phase has been prepared by surface radical chain‐transfer polymerization. The stationary phase was characterized by Fourier transform infrared spectrometry, thermogravimetric analysis, and element analysis. Fast and efficient separations of polar analytes, such as nucleosides and nucleic acid bases, water‐soluble vitamins and saponins, were well achieved in hydrophilic interaction chromatography mode. Additionally, a mixed mode of hydrophilic interaction and reversed‐phase could be also obtained in the analysis of polar and nonpolar compounds, including weak acidic phenols, basic anilines and positional isomers, with high resolution and molecular‐planarity selectivity, outperforming the commercially available amino column. Moreover, simultaneous separation of polar and nonpolar compounds was also achieved. In conclusion, the multimodal retention capabilities of the imidazolium‐embedded iodoacetamide‐functionalized silica‐based column could offer a wide range of retention behavior and flexible selectivity toward hydrophilic and hydrophobic compounds.     

Some insights on the description of gradient elution in reversed‐phase liquid chromatography

The so‐called “fundamental equation for gradient elution” has been used for modeling the retention in gradient elution. In this approach, the instantaneous retention factor (k) is expressed as a function of the change in the modifier content (φ(t_s)), t_s being the time the solute has spent in the stationary phase. This approach can only be applied at constant flow rate and with gradients where the elution strength depends on the column length following a f(t?l/u) function, u being the linear mobile phase flow rate, and l the distance from the column inlet to the location where the solute is at time t measured from the beginning of the gradient. These limitations can be solved by using the here called “general equation for gradient elution”, where k is expressed as a function of φ(t,l). However, this approach is more complex. In this work, a method that facilitates the integration of the “general equation” is described, which allows an approximate analytical solution with the quadratic retention model, improving the predictions offered by the “linear solvent strength model.” It also offers direct information about the changes in the instantaneous modifier content and retention factor, and gives a meaning to the gradient retention factor.     

Separation and purification of hydrolyzable tannin from Geranium wilfordii Maxim by reversed‐phase and normal‐phase high‐speed counter‐current chromatography

Three hydrolyzable tannins, geraniin, corilagin and gallic acid, main active components of Geranium wilfordii Maxim, have been separated and purified in one‐step by both reversed‐phase and normal‐phase high‐speed counter‐current chromatography. Gallic acid, corilagin and geraniin were purified from 70% aqueous acetone extract of G. wilfordii Maxim with solvent system n‐hexane–ethyl acetate–methanol–acetic acid–water (1:10:0.2:0.2:20) by reversed‐phase high‐speed counter‐current chromatography at purities of 94.2, 91.0 and 91.3%, at yields of 89.3, 82.9 and 91.7%, respectively. Gallic acid, corilagin and geraniin were purified with solvent system n‐hexane–ethyl acetate–methanol–acetic acid–water (0.2:10:2:1:5) by normal‐phase high‐speed counter‐current chromatography at purities of 85.9, 92.2 and 87.6%, at yields of 87.4, 94.6 and 94.3%, respectively. It was successful for both reversed‐phase and normal‐phase high‐speed counter‐current chromatography to separate high‐polarity of low‐molecular‐weight substances.     

Poly(l‐lactic acid)‐modified silica stationary phase for reversed‐phase and hydrophilic interaction liquid chromatography

Poly(l ‐lactic acid) is a linear aliphatic thermoplastic polyester that can be produced from renewable resources. A poly(l ‐lactic acid)‐modified silica stationary phase was newly prepared by amide bond reaction between amino groups on aminopropyl silica and carboxylic acid groups at the end of the poly(l ‐lactic acid) chain. The poly(l ‐lactic acid)‐silica column was characterized in reversed‐phase liquid chromatography and hydrophilic interaction liquid chromatography with the use of different mobile phase compositions. The poly(l ‐lactic acid)‐silica column was found to work in both modes, and the retention of test compounds depending on acetonitrile content exhibited “U‐shaped” curves, which was an indicator of reversed‐phase liquid chromatography/hydrophilic interaction liquid chromatography mixed‐mode retention behavior. In addition, carbonyl groups included into the poly(l ‐lactic acid) backbone work as an electron‐accepting group toward a polycyclic aromatic hydrocarbon and provide π–π interactions.     

Adsorption and recovery issues of recombinant monoclonal antibodies in reversed‐phase liquid chromatography†

The poor recovery of large biomolecules is a well‐known issue in reversed‐phase liquid chromatography. Several papers have reported this problem, but the reasons behind this behavior are not yet fully understood. In the present study, state‐of‐the‐art reversed‐phase wide‐pore stationary phases were used to evaluate the adsorption of therapeutic monoclonal antibodies. These biomolecules possess molar mass of approximately 150 000 g/mol and isoelectric points between 6.6 and 9.3. Two types of stationary phases were tested, the Phenomenex Aeris Widepore (silica based), with 3.6 μm superficially porous particles, and the Waters Acquity BEH300 (ethylene‐bridged hybrid), with 1.7 μm fully porous particles. A systematic investigation was carried out using 11 immunoglobulin G1, G2, and G4 antibodies, namely, panitumumab, natalizumab, cetuximab, bevacizumab, trastuzumab, rituximab, palivizumab, belimumab, adalimumab, denosumab, and ofatumumab. All are approved by the Food and Drug Administration and the European Medicines Agency in various therapeutic indications and are considered as reference antibodies. Several test proteins, such as human serum albumin, transferrin, apoferritin, ovalbumin, and others, possessing a molar mass between 42 000 and 443 000 g/mol were also evaluated to draw reliable conclusions. The purpose of this study was to find a correlation between the adsorption of monoclonal antibodies and their physicochemical properties. Therefore, the impact of isoelectric point, molar mass, protein glycosylation, and hydrophobicity was investigated. The adsorption of intact antibodies on the stationary phase was significantly higher than that of proteins of similar size, isoelectric point, or hydrophobicity. The present study also demonstrates the unique behavior of monoclonal antibodies, contributing some unwanted and unpredictable strong secondary interactions.     

Dicationic imidazolium ionic liquid modified silica as a novel reversed‐phase/anion‐exchange mixed‐mode stationary phase for high‐performance liquid chromatography

A dicationic imidazolium ionic liquid modified silica stationary phase was prepared and evaluated by reversed‐phase/anion‐exchange mixed‐mode chromatography. Model compounds (polycyclic aromatic hydrocarbons and anilines) were separated well on the column by reversed‐phase chromatography; inorganic anions (bromate, bromide, nitrate, iodide, and thiocyanate), and organic anions (p‐aminobenzoic acid, p‐anilinesulfonic acid, sodium benzoate, pathalic acid, and salicylic acid) were also separated individually by anion‐exchange chromatography. Based on the multiple sites of the stationary phase, the column could separate 14 solutes containing the above series of analytes in one run. The dicationic imidazolium ionic liquid modified silica can interact with hydrophobic analytes by the hydrophobic C₆ chain; it can enhance selectivity to aromatic compounds by imidazolium groups; and it also provided anion‐exchange and electrostatic interactions with ionic solutes. Compared with a monocationic ionic liquid functionalized stationary phase, the new stationary phase represented enhanced selectivity owing to more interaction sites.     

Evaluation and comparison of n‐alkyl chain and polar ligand bonded stationary phases for protein separation in reversed‐phase liquid chromatography

Protein retention is very sensitive to the change of solvent composition in reversed‐phase liquid chromatography for so called “on–off” mechanism, leading to difficulty in mobile phase optimization. In this study, a novel 3‐chloropropyl trichlorosilane ligand bonded column was prepared for protein separation. The differences in retention characteristics between the 3‐chloropropyl trichlorosilane ligand bonded column and n‐alkyl chain modified (C₂, C₄, C₈) stationary phases were elucidated by the retention equation . Retention parameters (a and c) of nine standard proteins with different molecular weights were calculated by using homemade software. Results showed that retention times of nine proteins were similar on four columns, but the 3‐chloropropyl trichlorosilane ligand bonded column obtained the lowest retention parameter values of larger proteins. It meant that their retention behavior affected by acetonitrile concentration would be different due to lower |c| values. More specifically, protein elution windows were broader, and retentions were less sensitive to the change of acetonitrile concentration on the 3‐chloropropyl trichlorosilane ligand bonded column than that on other columns. Meanwhile, the 3‐chloropropyl trichlorosilane ligand bonded column displayed distinctive selectivity for some proteins. Our results indicated that stationary phase with polar ligand provided potential solutions to the “on–off” problem and optimization in protein separation.     

Incorporation of ionic liquid into porous polymer monoliths to enhance the separation of small molecules in reversed‐phase high‐performance liquid chromatography

An ionic liquid was incorporated into the porous polymer monoliths to afford stationary phases with enhanced chromatographic performance for small molecules in reversed‐phase high‐performance liquid chromatography. The effect of the ionic liquid in the polymerization mixture on the performance of the monoliths was studied in detail. While monoliths without ionic liquid exhibited poor resolution and low efficiency, the addition of ionic liquid to the polymerization mixture provides highly increased resolution and high efficiency. The chromatographic performances of the monoliths were demonstrated by the separations of various small molecules including aromatic hydrocarbons, isomers, and homologues using a binary polar mobile phase. The present column efficiency reached 27 000 plates/m, which showed that the ionic liquid monoliths are alternative stationary phases in the separation of small molecules by high‐performance liquid chromatography     

Characterization and complete separation of major cyclolinopeptides in flaxseed oil by reversed‐phase chromatography

Organoleptic properties of flaxseed oil deteriorate during storage due to methionine oxidation in its major cyclolinopeptides. Cyclolinopeptide E was previously identified as being responsible for the manifestation of bitter taste with flaxseed oil ageing. We developed a chromatographic procedure to monitor the oxidation of major cyclic peptides in flaxseed oil. We also used liquid chromatography with mass spectrometry and high‐efficiency core–shell reversed‐phase sorbents to study the separation of cyclolinopeptides in detail. The Kinetex^TM family of stationary phases (C₈, C₁₈, phenyl‐hexyl) was tested, along with the standard porous Luna^TM C₁₈(2) media. We found that only the phenyl‐hexyl stationary phase allows for complete resolution of major cyclolinopeptides, thus permitting direct UV monitoring of degree of conversion for cyclolinopeptide B into C and L into E. We also report, for the first time, a significant effect of peak splitting for some methionine S‐oxide (Mso) containing cyclolinopeptides, which most likely appear due to diastereomerization. This results in poor separation efficiency for cyclolinopeptides F, G, and E, and gives baseline resolution of diastereomeric pairs for cyclolinopeptides I and P. Thus, a single oxidation of cyclolinopeptide N yields three distinct chromatographic peaks corresponding to cyclolinopeptide T (cyclo‐MsoLMPFFWV, reported for the first time) and pair of cyclolinopeptide I (cyclo‐MLMsoPFFWV) diastereomers.     

Reversed‐phase ion‐pair high‐performance liquid chromatography assay of polyprenyl diphosphate oligomer homologues

A reversed‐phase ion‐pair high‐performance liquid chromatography procedure was developed for the separation of polyprenyl diphosphate oligomer homologues obtained chemically from plant polyprenols. Tetrabutylammonium phosphate was used as the ion‐pair reagent, and the dependence of the separation quality on pH of ion‐pair reagent was investigated for the first time. The procedure is applicable for the control of commercial available polyprenyl monophosphates (the active components of veterinary drugs Phosprenyl and Gamapren) for the possible presence of polyprenyl diphosphate byproducts.     

Evaluation of electron capture detection in reversed‐phase HPLC for pharmaceutical analysis

The coupling of RP‐LC to electron capture detection (ECDNi⁶³) is described. To reduce the amount of mobile phase entering into the detector, interfacing was performed via a Scott‐type spray chamber. The performance of RP‐LC/ECDNi⁶³ was evaluated for pharmaceutical analysis and the results show that the system is able to work in a routine environment using columns with 2 mm id and common amounts of the organic modifiers methanol or ACN in the mobile phase. Because of the high sensitivity and selectivity toward electrophilic compounds, the use of this detector opens possibilities for the analysis of impurities down to the 0.05% level of active pharmaceutical ingredients (API).     

Purification of lignans from Fructus Arctii using off‐line two‐dimensional supercritical fluid chromatography/reversed‐phase liquid chromatography

As a common traditional Chinese medicine, Fructus Arctii has important clinical medical values. Its main components are lignans, which are difficult to separate and analyze because of the complex composition, similar chemical structures, and close properties. In this study, an off‐line two‐dimensional supercritical fluid chromatography/reversed‐phase liquid chromatography method, as well as an effective sample pretreatment method based on hydrophilic interaction chromatography material, was developed to enrich the minor lignan fractions and obtain high‐purity compounds. In total, 12 high‐purity compounds were isolated from Fructus Arctii . Their structures were identified by using high‐resolution mass spectrometry and nuclear magnetic resonance spectroscopy, which showed that all were lignans and that most of them were isomers. The results demonstrated the effective off‐line two‐dimensional supercritical fluid chromatography/reversed‐phase liquid chromatography method for the purification of lignans from Fructus Arctii . The separation protocol established here will be beneficial for the separation of complex samples from other kinds of natural products.     

Liquid chromatography of polyoxyethylenes under critical conditions: a thermodynamic study

Polyethylene glycols (PEG), fatty alcohol ethoxylates (FAE) and fatty acid polyglycol esters were analyzed by liquid chromatography under critical conditions (LCCC) on typical reversed phase columns in mobile phases consisting of methanol-water and acetone-water in the region of the critical adsorption point (CAP) for the oxyethylene unit. Critical conditions can be adjusted by varying the mobile phase composition or temperature. In methanol-water the temperature dependence is almost negligible, while it is much more pronounced in acetone-water. Critical conditions for the oxyethylene unit were realized on octadecyl phases in methanol-water containing 85.8 wt.% methanol and in acetone-water at 89.6 wt.% acetone. In the van't Hoff plots the logarithm of the distribution coefficient was plotted versus the reciprocal temperature. In all plots straight lines were found, from slope and intercept of which the entropy and enthalpy changes were calculated. The relation of the thermodynamic parameters -deltaG/RT, -deltaH/RT, and deltaS/R to the numbers of A and B chain units in block copolymers AB and BAB at the CAP for A is studied both theoretically and experimentally. Various approaches for obtaining the critical temperature are discussed.     

Hydrophilic interaction chromatography: A promising alternative to reversed‐phase liquid chromatography systems for the purification of small protonated bases

The overloaded band profiles of the protonated species of propranolol and amitriptyline were recorded under acidic conditions on four classes of stationary phases including a conventional silica/organic hybrid material in reversed‐phase liquid chromatography mode (BEH‐C₁₈), an electrostatic repulsion reversed‐phase liquid chromatography C₁₈ column (BEH‐C₁₈+), a poly(styrene‐divinylbenzene) monolithic column, and a hydrophilic interaction chromatography stationary phase (underivatized BEH). The same amounts of protonated bases per unit volume of stationary phase were injected in each column (16, 47, and 141 μg/cm³). The performance of the propranolol/amitriptyline purification was assessed on the basis of the asymmetry of the recorded band profiles and on the selectivity factor achieved. The results show that the separation performed under reversed‐phase liquid chromatography like conditions (with BEH‐C₁₈, BEH‐C₁₈+, and polymer monolith materials) provide the largest selectivity factors due to the difference in the hydrophobic character of the two compounds. However, they also provide the most distorted overloaded band profiles due to a too small loading capacity. Remarkably, symmetric band profiles were observed with the hydrophilic interaction chromatography column. The larger loading capacity of the hydrophilic interaction chromatography column is due to the accumulation of the protonated bases into the diffuse water layer formed at the surface of the polar adsorbent. This work encourages purifying ionizable compounds on hydrophilic interaction chromatography columns rather than on reversed‐phase liquid chromatography columns.     

Separation of imidacloprid and its degradation products using reversed phase liquid chromatography with water rich mobile phases

Water rich mobile phases in RPLC are not generally used because of the longer retention times involved when organic modifiers such as methanol or acetonitrile are used. The problem of longer retention times can be addressed using hydrophobic alcohols such as pentanol in low quantities (less than 1%) as organic modifiers. The advantages of using these mobile phases in RPLC for the separation of water soluble and weakly retained congeners is demonstrated through the separation of imidacloprid and its degradation products using a 0.4% pentanol in water mobile phase with 0.2% acetic acid.     

Separation behavior of basic compounds on unbonded silicon oxynitride and silica high‐performance liquid chromatography stationary phases with reversed‐phase eluents

Unbonded silicon oxynitride and silica high‐performance liquid chromatography stationary phases have been evaluated and compared for the separation of basic compounds of differing molecular weight, pK_a, and log D using aqueous/organic mobile phases. The influences of percentage of organic modifier, buffer pH, and concentration in the mobile phase on base retention were investigated on unbonded silicon oxynitride and silica phases. The results confirmed that unbonded silicon oxynitride and silica phases demonstrated excellent separation performance for model basic compounds and both the unbonded phases examined possessed a hydrophobic/adsorption and ion‐exchange character. The silicon oxynitride stationary phase exhibited high hydrophilicity compared with silica with a reversed‐phase mobile phase. An ion‐exclusion‐type mechanism becomes predominant for the separation of three aimed bases on the silicon oxynitride column at pH 2.8. Different from silicon oxynitride stationary phase, no obvious change for the retention time of three model bases on silica stationary phase at pH 2.8 can be observed.     

Temperature‐induced conformational transition of bovine serum albumin in neutral aqueous solution by reversed‐phase liquid chromatography

The temperature‐induced conformational transition of bovine serum albumin (BSA) in neutral aqueous solution was studied using intrinsic fluorescence emission spectrum, reversed‐phase liquid chromatography and sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and the conformation transition thermodynamic parameters were determined in the temperature range 12–50 °C. The results showed that, in the temperature range 12–20 °C, BSA only existed in a single conformation state A, while in the temperature range 22–50 °C, it existed in two different conformation states: A and B. The percentage of conformation state A decreased while that of conformation state B increased with the increase in temperatures, and when temperature approached 50 °C conformation state B accounted for approximately 25% of all conformation states of BSA. In the conformational transition of BSA from conformation state A to conformation state B, the positive enthalpy change, entropy change and free energy changes demonstrated that the conformational transition was endothermic, nonspontaneous and mainly entropy‐driven. © 2013 The Authors. Biomedical Chromatography published by John Wiley & Sons, Ltd.