Interaction Liquid Chromatography Stationary Phase Research Articles

Studies on the retention behavior of quercetin, phenol, and caffeine as test substances on selected neutral and charged hydrophilic interaction liquid chromatography stationary phases

AbstractThe influence of mobile phase composition and column temperature on the retention behavior of quercetin, phenol, and caffeine as test analytes was analyzed. The experiments were conducted in columns filled with the charged amine phase and the neutral amide phase. As mobile phases, the varying fractions of methanol or acetonitrile in water solutions were applied. The most likely retention mechanisms have been discussed. The results of investigations confirm the importance of a mixed adsorption‐partition retention mechanism in the hydrophilic interaction liquid chromatography process, regardless of the mobile phase composition and type of stationary phase. Investigations have also shown that in the tested systems the retention mechanism is also strongly column temperature‐dependent. Additionally, the efficiency of the tested columns was determined and analyzed as a function of the linear flow velocity. The obtained literature known as van Deemter plots confirms generally higher column efficiencies in the systems with acetonitrile as an organic component of the mobile phase. However, in some cases, the application of methanol allows obtaining similar column efficiencies. Furthermore, the results show that both columns tested exhibit comparable efficiency in the most tested systems and provide very good efficiency preservation over the wide range of eluent velocities.

Influence of different hydrophilic interaction liquid chromatography stationary phases on method performance for the determination of highly polar anionic pesticides in complex feed matrices.

Hydrophilic interaction liquid chromatography is an alternative liquid chromatography mode for separation of polar compounds. In the recent years, this liquid chromatography mode has been recognized as an important solution for the analysis of compounds not amenable to reverse phase chromatography. In this work, we evaluated three different hydrophilic liquid chromatography stationary phases for the determination of 14 highly polar anionic molecules including pesticides such as glyphosate, glufosinate, ethephon and fosetyl, their main metabolites, and bromide, chlorate, and perchlorate. Several mobile phase compositions were evaluated combined with different gradients for the chromatographic run. The two columns that presented the best results were used to assess the performance for the determination of the 14 compounds in challenging highly complex feed materials. Very different matrix effects were observed for most of the compounds in each column, suggesting that different interactions can occur. Using isotopically labeled internal standards, acceptable quantitative performance and identification could be achieved down to 0.02 mg kg−1 (the lowest level tested) for most compounds. While one column was found to be favorable in terms of scope (suited for all 14 compounds), the other one was more suited for quantification and identification at lower levels, however, not for all analytes tested.

An alternative approach for the preparation of a core-shell bimetallic central metal-organic framework as a hydrophilic interaction liquid chromatography stationary phase.

A new type of core-shell composite material was prepared and applied as a hydrophilic interaction liquid chromatography (HILIC) stationary phase. In this work, silica spheres were first modified with a bimetallic central metal-organic framework (ZnCoMOF) by a new strategy of static self-assembled in situ growth. This strategy was beneficial for increasing the electrostatic interaction between the MOF ligand and silica via introducing a sodium dodecylbenzenesulfonate (SDBS) group. The ZnCoMOF@silica stationary phase was characterized and evaluated in comparison with amino-modified and bare silica columns in terms of various polar analytes including eight nucleosides and nucleobases, seven carbohydrates, and multiple sulfonamides and antibiotics. The effects of organic solvent concentration, water content, the concentration of the salt and the pH of the buffer solution on the retention time were studied, which demonstrated the typical retention behavior of HILIC on the ZnCoMOF@silica column. Compared with most reported MOF-based stationary phases, the new composite material showed excellent hydrophilic properties and separation efficiency for various polar analytes. Moreover, the obtained stationary phase showed good reproducibility and stability. The relative standard deviation (RSD) of the retention time for repeatability was found to range from 0.1% to 0.6%, and the RSD of the retention time for stability was found to range from 0.3% to 0.7%. Furthermore, the column batch-to-batch reproducibility showed excellent preparation reproducibility, which few reported in most previous MOF@silica composite materials. This specific preparation method offers an easy and novel way to manipulate the amount of MOF particles on silica, which extends a universal way to produce various MOF@silica stationary phases by the method of static self-assembled in situ growth.

Development and Analytical Validation of the Methodology for Vitamins in Tablets by Ultra-Performance Liquid Chromatography.

In the present study, we developed a reliable and robust chromatographic method for the quantification of multivitamins in tablet samples by ultra-performance liquid chromatography (UPLC) with photodiode array detection. The vitamins nicotinamide, pyridoxine, riboflavin, and thiamin were analyzed and quantified in a total analysis time of 2.5minutes, using hydrophilic interaction liquid chromatography stationary phase. Tocopherol acetate and cyanocobalamin were analyzed and quantified in a total analysis time of 2.5minutes, using reversed-phase (RP)-UPLC. The analysis time reported here is lower than that of similar methods reported in the literature for single vitamin determination. The method linearity exhibits a good correlation coefficient (R2=0.998) with the relative residual standard deviation in the acceptable limit of 2.0%. The developed methods were validated, and the results demonstrated that the proposed analytical method showed to be selective, sensitive, accurate, and robust for the quantification of evaluated vitamins in multivitamin tablets. The work was fully developed in the quality control laboratory of a pharmaceutical industry in the Agroindustrial District of Anápolis (DAIA, Goiás, Brazil), where the product is manufactured.

Comparison of underivatized silica and zwitterionic sulfobetaine hydrophilic interaction liquid chromatography stationary phases for global metabolomics of human plasma

To increase metabolome coverage in global LC–MS metabolomics, often both reversed-phase liquid chromatography (RPLC) and hydrophilic-interaction liquid chromatography (HILIC) are implemented in parallel. However, there is a lack of consensus in the literature on the best HILIC stationary phase to employ for global metabolomics of human biological fluids. The objective of this study was to compare in detail the performance of two commonly employed HILIC phases: zwitterionic sulfobetaine ZIC-HILIC stationary phase and an underivatized silica HILIC stationary phase. During method development, the effect of salt concentration in the mobile phase was also investigated, and 5 mM ammonium acetate was selected. The stationary phases were evaluated using a mixture of 37 polar standards covering a range of logP values (-10 to 3.73), molecular weights (59–776 Da), charges (15 anions, 11 cations, and 11 neutral) as well as 17 lipid standards to understand phospholipid behaviour on the two stationary phases. The criteria used for the comparison included the quality of the chromatographic peak shape, adequate analyte retention, peak separation capability, and metabolite coverage. The zwitterionic ZIC-HILIC column provided better chromatographic performance over the silica stationary phase with 14 standards achieving good quality peaks compared to the 7 with the silica column. Only 2 standards were undetected with the ZIC-HILIC column compared to the 14 undetected with the silica column. In human plasma, 1966 and 1650 metabolites were observed on the ZIC-HILIC column in positive and negative electrospray ionization (ESI) respectively. On the silica HILIC column, 1773 and 2028 metabolites were observed in positive and negative ESI respectively, showing comparable performance of the two phases. Next, the effect of adding 10 mM ammonium phosphate to the samples to improve the analyte peak shape and metabolite coverage was investigated for both ZIC-HILIC and silica HILIC. In contrast with recently reported results for pZIC-HILIC, there was no clear evidence that ammonium phosphate addition was beneficial for human plasma samples. In conclusion, ZIC-HILIC provided better chromatographic performance for polar plasma metabolomics than underivatized silica in terms of chromatographic peak shape and chromatographic resolution, while maintaining comparable metabolite coverage. The addition of ammonium phosphate to human plasma was not beneficial for either of the two stationary phases.

Quantification of Theophylline in Guinea Pig Plasma by LC-MS/MS Using Hydrophilic Interaction Liquid Chromatography Stationary Phase: Method Development, Validation, and Application in Study

AbstractTheophylline has been used in the treatment of bronchial asthma and chronic obstructive pulmonary disease (COPD) for over 70 years. In order to maximize the effectiveness and safety of theophylline therapy it is important to individualize the dosage of the drug. In our study we focused on determination of theophylline concentrations in guinea pig plasma. A rapid, specific, and reliable LC-MS/MS-based method was developed and validated according to European Medicine Agency (EMA) guidelines. A hydrophilic interaction liquid chromatography (HILIC) separation mode for reduction time of sample preparation was used. The analysed sample was quantified in a positive ionization mode. Multiple reaction monitoring (MRM) using transition m/z 181.06→124.06 and m/z 187.17→127.06 was performed to quantify theophylline with deuterated internal standard ([2H6]-theophylline), respectively. Modification of collision energies was performed in parallel with chromatographic separation to further eliminate interference from the matrix. The method was validated for a range of 0.5 to 30 μg/mL of plasma sample. The intra-day and inter-day precision and accuracy of the quality control samples at low, me dium, and high concentration levels exhibited relative standard deviations (RSD) of less than 10 %. The method was successfully applied for the quantitation of theophylline in guinea pig plasma for better understanding its effects in a model of ovalbumin-induced allergic inflammation.

Modelling of Hydrophilic Interaction Liquid Chromatography Stationary Phases Using Chemometric Approaches.

Metabolomics is a powerful and widely used approach that aims to screen endogenous small molecules (metabolites) of different families present in biological samples. The large variety of compounds to be determined and their wide diversity of physical and chemical properties have promoted the development of different types of hydrophilic interaction liquid chromatography (HILIC) stationary phases. However, the selection of the most suitable HILIC stationary phase is not straightforward. In this work, four different HILIC stationary phases have been compared to evaluate their potential application for the analysis of a complex mixture of metabolites, a situation similar to that found in non-targeted metabolomics studies. The obtained chromatographic data were analyzed by different chemometric methods to explore the behavior of the considered stationary phases. ANOVA-simultaneous component analysis (ASCA), principal component analysis (PCA) and partial least squares regression (PLS) were used to explore the experimental factors affecting the stationary phase performance, the main similarities and differences among chromatographic conditions used (stationary phase and pH) and the molecular descriptors most useful to understand the behavior of each stationary phase.

Evaluation of hydrophilic interaction liquid chromatography stationary phases for analysis of opium alkaloids

The separation of a mixture containing five major opium alkaloids, namely morphine, codeine, thebaine, noscapine and papaverine has been investigated in hydrophilic interaction liquid chromatography (HILIC) mode using five different stationary phases: bare silica, zwitterion, aminopropyl, diol and cyanopropyl. In order to propose the appropriate column for separation and purification, retention behaviors of the five natural opioids have been studied on mentioned HILIC stationary phases. The mechanism of separation in diverse HILIC media, based on the formation of water-rich layer on surface of the HILIC stationary phases and the physicochemical properties of opium alkaloids, such as pKa (acidic pK) and the octanol-water distribution coefficient (log Do/w) are discussed. Chromatographic responses including modified limit of detection LODm, signal to noise ratio (S/N)m, and defined modified RSm have considered for suggestion of the suitable column for quantitative/qualitative and preparative purposes. According to the obtained results, diol stationary phase is best suited for analytical chromatography, whereas bare silica and zwitterionic stationary phases are appropriate for preparative applications.

Chemometric evaluation of hydrophilic interaction liquid chromatography stationary phases: resolving complex mixtures of metabolites

Different hydrophilic interaction liquid chromatography (HILIC) stationary phases have been evaluated using different chemometric methods with the aim of their application in metabolomics studies.

Preparation, evaluation, and application of a novel reversed-phase/zwitterionic/hydrophilic interaction liquid chromatographic mixed-mode stationary phase

ABSTRACTThe present study described the preparation and application of a reversed-phase/zwitterionic/hydrophilic interaction liquid chromatography stationary phase, named as SIL-PS. The SIL-PS was prepared through a four-step reaction, chemical bonding, nucleophilic addition, SN1 substitution, and sulfonation on the silica matrix. It was featured with C12 alkyl chain, quaternary ammonium, tertiary amine, and sulfonate groups. After SIL-PS was packed into the stainless steel column (150 × 2.1 mm i.d.), chromatographic parameters, including acetonitrile content, pH, and ionic strength of the mobile phase, and the column temperature, were systematically investigated to study the retention mechanism. Electrostatic adsorptive/repulsive, partition, and hydrogen-bonding interactions were demonstrated to contribute to the retention. The stability of the SIL-PS was satisfactory, with relative standard deviations of retention factors of 1.93, 2.08, and 1.90% for loxoprofen, adenosine, and liquiritin, respectively. Additionally, to investigate the separation selectivity, the non-steroidal anti-inflammatory drugs, nucleobases/nucleotides, and alkaloids/glycosides were separated; the HPLC fingerprinting of the Cortex phellodendri extract was also conducted, and the separation performance was superior to that of the C18 column in terms of peak shape, resolution, and analytical time. The results revealed that the prepared SIL-PS possessed multifunctionalities for multiretention and could be promising for complicated samples.

Silica-based 2-(N,N-dimethylamino)-1,3-propanediol hydrophilic interaction liquid chromatography stationary phase for separating cephalosporins and carbapenems.

A silica-based stationary phase bearing both hydrophilic hydroxyl and amino groups was developed by covalently bonding a small molecular N,N-dimethylamino 1,3-propanediol moiety onto silica beads via copper(I)-catalyzed Huisgen azide-alkyne 1,3-dipolar cycloaddition (CuAAC). This new stationary phase showed good HILIC characteristics and high column efficiency (the theoretical plate number is up to 37000platesm(-1) in the case of inosine) in the separation of polar compounds, such as nucleosides and bases, organic acids, cephalosporins, and carbapenems.

Cucurbit(6)uril immobilized on silica: a novel high-performance liquid chromatographic stationary phase.

In the present study, one of the new generation of host molecules, cucurbit(6)uril (CB(6)), was immobilized onto silica (CB(6)/SiO2 ) by a sol-gel approach. CB(6)/SiO2 was characterized by NMR spectroscopy, Fourier transform infrared spectroscopy, thermogravimetric analysis, and elemental analysis. It was used as a high-performance liquid chromatographic stationary phase and its chromatographic performance was systematically investigated with different types of analytes as probes. The results revealed that the CB(6)/SiO2 stationary phase exhibited weak hydrophobic and strong hydrophilic properties. Hence, the variables for hydrophilic interaction liquid chromatography, including components and pH of the mobile phase, were further investigated to explore the retention mechanism of this CB(6)/SiO2 stationary phase. For less polar analytes, both hydrophobic and hydrophilic interactions could contribute to the retention, while for polar analytes, hydrophilic interaction may be predominant. Compared to the tetraethoxylsilane-coated SiO2 stationary phases, the CB(6)/SiO2 stationary phase exhibited a different retention behavior toward basic analytes with excellent stability. It is a novel promising hydrophilic interaction liquid chromatography stationary phase.

Speciation and Isotope Dilution Analysis of Gadolinium-Based Contrast Agents in Wastewater

The fate of Gadolinium (Gd)-based contrast agents for magnetic resonance imaging (MRI) during sewage treatment was investigated. The total concentration of Gd in influent and effluent 2 and 24 h composite samples was determined by means of isotope dilution analysis. The balancing of Gd input and output of a sewage plant over seven days indicated that approximately 10% of the Gd is removed during treatment. Batch experiments simulating the aeration tank of a sewage treatment plant confirmed the Gd complex removal during activated sludge treatment. For speciation analysis of the Gd complexes in wastewater samples, high performance liquid chromatography (HPLC) was hyphenated to inductively coupled plasma sector field mass spectrometry (ICP-SFMS). Separation of the five predominantly used contrast agents was carried out on a new hydrophilic interaction liquid chromatography stationary phase in less than 15 min. A limit of detection (LOD) of 0.13 μg/L and a limit of quantification of 0.43 μg/L could be achieved for the Gd chelates without having to apply enrichment techniques. Speciation analysis of the 24 h composite samples revealed that 80% of the Gd complexes are present as Gd-BT-DO3A in the sampled treatment plant. The day-of-week dependent variation of the complex load followed the variation of the total Gd load, indicating a similar behavior. The analysis of sewage sludge did not prove the presence of anthropogenic Gd. However, in the effluent of the chamber filter press, which was used for sludge dewatering, two of the contrast agents and three other unknown Gd species were observed. This indicates that species transformation took place during anaerobic sludge treatment.

Effect of silica gel modification with cyclofructans on properties of hydrophilic interaction liquid chromatography stationary phases

Hydrophilic interaction liquid chromatography (HILIC) offers very good possibilities for separation of polar compounds as an alternative to reversed phase HPLC where polar compounds are not sufficiently retained. HILIC is becoming more popular for the analysis of biologically interesting (active) analytes. Various stationary phases are commercially available however, development of new materials (sorbents) suitable for HILIC systems still continues. Silica gel columns can be used directly but their modification can improve separation ability of the stationary phases. Cyclofructan-based stationary phases are demonstrated as possible HILIC columns in this work. The effect of silica gel modification by cyclofructan and a derivatized cyclofructan was studied in detail. HILIC separation systems with silica gel, cyclofructan and isopropyl cyclofructan modified silica stationary phases were compared. The detailed study of chromatographic behavior of peptides revealed that multimodal retention mechanism is present in systems with these stationary phases. Mobile phase composition changes the types of interactions and their strengths. It appears that ability to donate protons and dispersion forces are the main interactions that affect retention in HILIC with cyclofructan-based columns while they are less important in separation systems with bare silica stationary phase. Suitability of cyclofructan-based stationary phases in HILIC for separation of pentapeptides and nonapeptides was demonstrated.

Monolithic poly (SPE-co-BVPE) capillary columns as a novel hydrophilic interaction liquid chromatography stationary phase for the separation of polar analytes

A novel hydrophilic interaction liquid chromatography (HILIC) stationary phase was prepared by the co-polymerisation of zwitterionic N,N′-dimethyl-N-methacryloxyethyl-N-(3-sulfopropyl) ammonium betaine (SPE) and the crosslinker 1,2-bis(p-vinylphenyl) ethane (BVPE) in the presence of the porogens, toluene and methanol. Monolithic columns were produced by carrying out the α,α'-azoisobutyronitrile (AIBN) initiated reaction for 1, 2, 4, 8 and 12h inside a 200μm i.d. fused silica capillary at 75°C (water bath). The optimum polymerisation time was shown to be 2h, as this resulted in good porosity, due to enlarged flow-channels and the presence of a higher proportion of mesopores provided a relatively larger surface area than the other columns. The chromatographic properties of the optimised poly (SPE-co-BVPE) monolithic column were evaluated with test mixtures containing both basic and neutral compounds in the HILIC gradient separation mode. This produced relatively sharp peaks (average peak width at half height=0.1min) with average asymmetry factors of 1.4 and baseline resolution was obtained for all the compounds. Using the isocratic separation of the test mixture, the number of theoretical plates (N) per metre calculated was between 26,888 and 35,930 by using average values obtained for triplicate injections of the compounds thiourea, toluene and acrylamide.

Preparation and chromatographic evaluation of a cysteine-bonded zwitterionic hydrophilic interaction liquid chromatography stationary phase

A cysteine-bonded zwitterionic hydrophilic interaction chromatography (HILIC) stationary phase (Click TE-Cys) was prepared based on the “thiol-ene” click chemistry. The Click TE-Cys material was characterized by solid state 13C cross polarization/magic-angle spinning (CP/MAS) NMR and elemental analysis. The dynamic evaluation for cytosine, cytidine and orotic acid was performed using Van Deemter plots. The plate height values were no more than 24μm for the flow rate between 0.5 and 5.4mms−1 (0.3–3.5mLmin−1), which proved the excellent separation efficiency of Click TE-Cys stationary phase. The influences of the content of water, concentration of salt and pH of the buffer solution on the retention of model compounds were investigated. The results demonstrated that the separation of polar analytes was dominated by the partitioning mechanism, while the contribution of electrostatic interaction was minor. The thermodynamic characteristic of Click TE-Cys stationary phase was also studied according to van’t Hoff plot. An exothermic process for transferring analytes from the mobile phase to the stationary phase was observed and a linear relationship for lnk and 1/T was achieved, indicating no change of retention mechanism within the measured temperature range. Besides, the zwitterionic stationary phase exhibited good stability. Considering the high hydrophilicity of Click TE-Cys stationary phase, the application in the separation of protein tryptic digests was carried out using hydrophilic interaction chromatography–electrospray ionization mass spectrometry (HILIC–ESI-MS). More peaks were adequately resolved on the Click TE-Cys column comparing with that on the TSK Amide-80 column. In addition, the orthogonality between HILIC and RPLC system was investigated utilizing geometric approach. The XTerra MS C18 and Click TE-Cys column displayed great difference in separation selectivity, with the orthogonality reaching 88.0%. On the other hand, the orthogonality between Click TE-Cys and TSK Amide-80 system was 21.4%, i.e. the selectivity was similar but slightly different from each other. The successful separation of protein digests indicated the great potential of Click TE-Cys stationary phase in the separation of complex samples and applicability in two-dimensional liquid chromatography (2D-LC).

Development and evaluation of new zwitterionic Hydrophilic interaction liquid chromatography stationary phases based on 3-P,P-diphenylphosphonium-propylsulfonate

New zwitterionic stationary phases were synthesized by covalently bonding 3-P,P-diphenylphosphonium-propylsulfonate to silica gel. The resulting materials possess both a negatively charged sulfonate group and a positively charged quaternary phosphonium group, which means that there is no net charge over a wide pH range. The retention mechanism and chromatographic behavior of polar solutes under HILIC conditions were studied on these zwitterionic phases. Compared to the commercial ZIC-HILIC column and a bare silica gel stationary phase, the newly synthesized zwitterionic stationary phases provided greater retention, higher peak efficiency and better peak symmetry in the HILIC mode. The analytes examined included: β-blockers, nucleic acid bases and nucleosides, salicylic acid and its analogues, and water soluble vitamins. Factors, such as the type of organic modifiers, solvent composition, pH and the buffer concentration of the mobile phase, have been considered as potential variables for controlling the chromatographic retention of polar analytes.

Chromatographic characterization of hydrophilic interaction liquid chromatography stationary phases: Hydrophilicity, charge effects, structural selectivity, and separation efficiency

Fourteen commercially available particle-packed columns and a monolithic column for hydrophilic interaction liquid chromatography (HILIC) were characterized in terms of the degree of hydrophilicity, the selectivity for hydrophilic–hydrophobic substituents, the selectivity for the regio and configurational differences in hydrophilic substituents, the selectivity for molecular shapes, the evaluation of electrostatic interactions, and the evaluation of the acidic–basic nature of the stationary phases using nucleoside derivatives, phenyl glucoside derivatives, xanthine derivatives, sodium p-toluenesulfonate, and trimethylphenylammonium chloride as a set of samples. Principal component analysis based on the data of retention factors could separate three clusters of the HILIC phases. The column efficiency and the peak asymmetry factors were also discussed. These data on the selectivity for partial structural differences were summarized as radar-shaped diagrams. This method of column characterization is helpful to classify HILIC stationary phases on the basis of their chromatographic properties, and to choose better columns for targets to be separated. Judging from the retention factor for uridine, these HILIC columns could be separated into two groups: strongly retentive and weakly retentive stationary phases. Among the strongly retentive stationary phases, zwitterionic and amide functionalities were found to be the most selective on the basis of partial structural differences. The hydroxyethyl-type stationary phase showed the highest retention factor, but with low separation efficiency. Weakly retentive stationary phases generally showed lower selectivity for partial structural differences.

Determination of melamine in milk powder using zwitterionic HILIC stationary phase with UV detection

A hydrophilic interaction liquid chromatography-UV method was developed for the determination of melamine in milk powder. Sulfobetaine type zwitterionic hydrophilic interaction liquid chromatography stationary phase was used to achieve straightforward separation of melamine in milk powder without any sample derivatization or addition of ion-pair reagent. The sample preparation was simple and fast with the steps of acetonitrile/perchloric acid extraction-centrifugation-filtration. No SPE or other pre-concentration procedure was required. By using large volume sample injection and choosing low UV wavelength (210 nm), the LOD and LOQ were 0.005 and 0.015 microg/mL for melamine standards, and 0.02 and 0.06 microg/mL for the spiked milk extracts. The LOD and LOQ of the latter correspond to 0.95 and 2.2 microg/g melamine in the milk powder. The correlation coefficients for melamine standard, pre-spiked milk extracts and post-spiked milk extracts in the range of 0-0.5 microg/mL were 0.9978, 0.9976 and 0.9995, respectively. This newly developed method is sensitive and cost effective, therefore, suitable for screening of melamine in tainted milk products.