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.     

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.     

Comparison of retention on traditional alkyl, polar endcapped, and polar embedded group stationary phases

The development of new RP stationary phases containing polar groups has provided the chromatographer with a variety of stationary phase choices with differing selectivities. Polar endcapped and polar embedded group stationary phases have found use in solving a wide variety of separation problems, especially for the efficient separation of organic bases as well as separations necessitating the use of highly aqueous mobile phases. In this report, the retention thermodynamics of small, nonpolar solutes on traditional alkyl, polar endcapped, and polar embedded group stationary phases are compared. It is found that the nonpolar (methylene) transfer enthalpy is less favorable when polar embedded group phases are used, when compared to traditional or polar endcapped phases. In contrast, the transfer enthalpy of a phenyl group is found to be more favorable when a polar endcapped phase is used. In addition, the retention characteristics of these phases are compared using a set of solutes with differing solvatochromic parameters. Hydrogen-bond acids appear to have enhanced retention on polar embedded group phases, while hydrogen-bond bases have enhanced retention on polar endcapped phases.     

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.     

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.     

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.     

Void volume determination and experimental probes in reversed phase chromatography

Summary In reversed-phase liquid chromatography with n-alkyl bonded silica, the dead volume (V₀) of the column is theoretically indeterminate owing to adsorption of organic modifier on n-alkyl chains and of water on silanol groups. With binary mobile phases, retention volumes of the mobile phase components and of their deuterated species are relaeed to the adsorption isotherms and V₀ by equations which can be solved with some assumptions on the adsorbed layer composition. Methanol-water and acetonitrile-water systems are studied. As the experimental excess isotherm shows a linear part in the concentration range 50–80% in organic modifier, the hypothesis of an adsorbed layer of constant composition in this range is possible. When increasing the water content of the mobile phase, adsorption of water occurs up to saturation of silanol groups. Then the assumption of a constant water content for a mobile phase having more than 50% of water is applied. With the hypothesis of a constant adsorbed content of organic modifier when the eluent has more than 80% of organic modifier, V₀ and the absolute isotherms are calculated over the entire range of mobile phase composition. Experimental retention behavior of the mobile phase components are totally explained by these V₀ determinations. The retention times of commonly used V₀ markers are compared with V₀ values. It is shown that, when buffering the eluent, no visible effect on the distribution equilibrium is observed, so that injection of concentrated potassium nitrate is a convenient method to measure V₀. With a few solutes with are UV detectable it is possible to measure V₀ whatever the mobile phase composition in methanol-water and acetonitrile-water systems.     

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.     

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).     

HPLC with polysaccharide chiral stationary phase in polar‐organic phase mode: Application to the asymmetric epoxidation of allylic alcohols

A simple and rapid HPLC method using a polysaccharide‐based chiral stationary phase (Chiralpak AD‐H) in polar‐organic phase mode has been developed for direct resolution of glycidyl nitrobenzoate (GNB) and 2‐methyl glycidyl nitrobenzoate (MGNB) enantiomers. ACN and methanol were used as mobile phase and the effects of the addition of ethanol and 2‐propanol as organic modifier in the mobile phase, flow rate and the column temperature were tested. The optimized conditions were: methanol/ethanol (80:20) at a flow rate of 0.9 mL/min and 40°C. Analysis time was ?13 min and the chiral resolution was ?2. The method was validated and resulted to be selective, precise and accurate. The method was found to be linear in 2–300 μg/mL range (R² >0.999) with an LOD nearly 0.5 μg/mL for four enantiomers. GNB and MGNB enantiomers were obtained by asymmetric epoxidation of allyl alcohol and 2‐methyl allyl alcohol, respectively, using chiral titanium–tartrate complexes as catalyst and dichloromethane as solvent after in situ derivatization of the intermediate glycidols derivatives. The quite simple and rapid validated method was applied successfully for direct determination of the enantiomeric excess (?90%) and yield obtained in real samples of asymmetric epoxidation of allylic alcohols without further purification, workup or solvent removal. The method provides a useful and value‐added tool for controlling the enantiomeric purity of the synthesized epoxides.     

Preparation of a stationary phase with s‐triazine ring embedded group for reversed phase high‐performance LC

This paper describes the synthesis and chromatographic evaluation of a new polar‐embedded stationary phase, which utilized 2,4,6‐trichloro‐1,3,5‐triazine as the spacer. The resulting materials were characterized by elemental analysis, IR, and solid‐state ¹³C NMR. Empirical test mixtures were utilized to evaluate the column, and showed that it had good performance for basic compounds and high selectivity for polyaromatic hydrocarbons. Moreover, the novel stationary phase has unique property, especially in the separation of “homologous alkaloids” from natural products.     

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.     

Mobile phase additives for enhancing the chromatographic performance of astaxanthin on nonendcapped polymeric C30‐bonded stationary phases

Astaxanthin shows peak deformation and reduced peak area response when eluted with methanol and methyl tert‐butyl ether on nonendcapped polymeric C₃₀‐bonded HPLC phases. The present study tested different column manufacturers, column batches, and ten mobile phase additives including acids, bases, buffers, complexing and antioxidant agents for improvement of peak shape and peak area response. Concerning chromatographic benefits and feasibility, ammonium acetate was found to be the best additive followed by triethylamine for all columns tested. Variation of the mobile phase pH equivalent and the column temperature showed no synergistic effects on peak shape and peak area response. Results indicate that peak tailing and variation of peak area response are due to different on‐column effects. Possible mechanisms of the observed phenomenon will be discussed.     

Phenyl‐bonded stationary phases—The influence of polar functional groups on retention and selectivity in reversed‐phase liquid chromatography

The chromatographic properties of four phenyl‐bonded phases with different structures were studied. The columns used were packed with a stationary phase containing a phenyl ring attached to the silica surface using different types of linkage molecules. As a basic characteristic of the bonded phases, the hydrophobicity and silanol activity (polarity) were investigated. The presence of the polar amino and amide groups in the structure of the bonded ligand strongly influences the polarity of the bonded phase. Columns were compared according to methylene selectivity using a series of benzene homologues and according to their shape and size selectivity using polycyclic aromatic hydrocarbons. The measurements were done using methanol/water and acetonitrile/water mobile phases. The presented results show that the presence of polar functional groups in the ligand structure strongly influences the chromatographic properties of the bonded phase.     

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.     

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.     

Separation of xanthines in hydro‐organic and polar‐organic elution modes on a titania stationary phase

Hydrophilic interaction LC was investigated in hydro‐organic and nonaqueous elution modes on a titania column by using a set of N‐methyl xanthines as neutral polar probes. To get information regarding the mechanisms that are behind the discrimination of these analytes in hydrophilic interaction, we focused our study on the type and amount of organic modifier as a critical yet rarely explored mobile phase parameter. Several alcohols such as methanol, ethanol, and isopropanol were studied as substitutes to acetonitrile in hydro‐organic elution mode. Compared to silica, the investigation of the eluotropic series of these alcohols on titania highlighted a different implication in the retention mechanism of the xanthine derivatives. At low amounts of protic solvents, the adsorption mainly characterized the retention of analytes on bare silica; whereas mixed interactions including adsorption and ligand exchange were identified on native titania. To investigate the peculiar behavior of alcohols on the metal oxide, methanol, ethanol, and ethylene glycol were tested in replacement of water in polar‐organic elution mode. Distinctive effects on the chromatographic retention and selectivity of xanthines were noticed for the dihydric alcohol, which was found to be a stronger eluting component than water on titania.     

Separation of polyethylene glycols and maleimide‐terminated polyethylene glycols by reversed‐phase liquid chromatography under critical conditions

The separation of polyethylene glycols and maleimide‐substituted polyethylene glycol derivatives based on the number of maleimide end‐groups under critical liquid chromatography conditions has been investigated on a reversed‐phase column. The critical solvent compositions for nonfunctional polyethylene glycols and bifunctional maleimide‐substituted polyethylene glycols were determined to be identical at about 40% acetonitrile in water on a reversed‐phase octadecyl carbon chain‐bonded silica column using mixtures of acetonitrile and water of varying composition as the mobile phase at 25°C. The maleimide‐functionalized polyethylene glycols were successfully separated according to maleimide functionality (with zero, one, two, or three maleimide end‐groups, respectively) under the critical isocratic elution conditions without obvious effect of molar mass. The separation was mainly due to the hydrophobic interaction between the maleimide end‐groups and the column packing. Off‐line matrix‐assisted laser desorption/ionization time of flight mass spectrometry was used to identify the repeating units and, especially, the end‐groups of the maleimide‐substituted polyethylene glycols. Liquid chromatography analysis at critical conditions could provide useful information to optimize the synthesis of functional polyethylene glycols. To our knowledge, this is the first report of the baseline separation of maleimide‐functionalized polyethylene glycols based on the functionality independent of the molar mass without derivatization by isocratic elution.     

Protonation of polyaniline‐coated silica stationary phase affects the retention behavior of neutral hydrophobic solutes in reversed‐phase capillary liquid chromatography

Because of its high conductivity when acid doped, polyaniline is known as a synthetic metal and is used in a wide range of applications, such as supercapacitors, biosensors, electrochromic devices, or solar and fuel cells. Emeraldine is the partly oxidized, stable form of polyaniline, consisting of alternating diaminobenzenoid and iminoquinoid segments. When acidified, the nitrogen atoms of emeraldine become protonated. Due to electrostatic repulsion between positive charges, the polarity and morphology of emeraldine chains presumably change; however, the protonation effects on emeraldine have not yet been clarified. Thus, we investigated these changes by reversed‐phase capillary liquid chromatography using a linear solvation energy relationship approach to assess differences in dominant retention interactions under a significantly varied mobile phase pH. We observed that hydrophobicity dominates the intermolecular interactions under both acidic and alkaline eluent conditions, albeit to different extents. Therefore, by tuning the mobile phase pH, we can even modulate the retention of neutral hydrophobic solutes, such as aromatic hydrocarbons, because the pH‐dependent charge and structure of polymer chains of the emeraldine‐coated silica stationary phase show a mixed‐mode separation mechanism.     

Retention process in reversed phase TLC systems with polar bonded stationary phases

The retention of a solute in RP chromatography is a very complex process which depends on many factors. Therefore, the study of the influence of a mobile phase modifier concentration on the retention in different reversed phase chromatographic systems is very important for understanding the rules governing retention and mechanisms of substance separation in a chromatographic process. Composition changes and the nature of mobile phases enable tuning of the separated analytes' retention over a wide range of retention parameters and optimization of the chromatographic process as well. Optimization of the chromatographic process can be achieved by several different methods; one of them is the so-called interpretative strategy. The key approach adopted in this strategy is the implementation of adequate retention models that couple the retention of solute with the composition of a mixed mobile phase. The use of chemically bonded stationary phases composed of partially non-bonded silica matrix and organic ligands bonded to its surface in everyday chromatography practice leads to questions of the correct definition of the retention model and the dominant retention mechanism in such chromatographic systems. The retention model for an accurate prediction of retention factor as a function of modifier concentration and the heterogeneity of the adsorbent surface should be taken into consideration. In this work the influence of mobile-phase composition on the retention of sixteen model substances such as phenols, quinolines, and anilines used as test analytes in different RP-TLC systems with CN-, NH2-, and Diol-silica polar bonded stationary phases has been studied. The aim of this study is to compare the performance of three valuable retention models assumed as the partition, adsorption/partition, and adsorption mechanism of retention. All the models were verified for different RP-TLC systems by three statistical criteria. The results of investigations presented in this work demonstrate that the best agreement between the experimental and calculated Rf values was obtained by the use of new-generation retention models, which assume heterogeneity of adsorbent surface. The results reported here show that heterogeneity of the adsorbent surface may be important in analysis of the elution process in liquid chromatography. Consideration of the goodness of fit for the experimental data to the examined retention models is in conformity with the adsorption mechanism of retention on all polar bonded stationary phases in most eluent systems for most investigated compounds.