Water-rich Mobile Phases Research Articles

Per-Aqueous Liquid Chromatography for the Separation of Polar Compounds-Preparation and Characterization of Poly(ethylene oxide-co-dimethylsiloxane) Based Stationary Phase

Poly(ethylene oxide-co-dimethylsiloxane) was thermally immobilized on silica particles- Si(PEO)-to separate small polar compounds with water-rich mobile phases. Poly(ethylene oxideco- dimethylsiloxane) content on Si(PEO) stationary phase was optimized using a central composite design. Infrared spectroscopy, scanning electron microscopy, and thermogravimetric analysis morphologically and structurally characterized the optimized material. Separation of standard test mixtures showed that the Si(PEO) phase had a typical reversed-phase elution order. However, the Si(PEO) phase retained polar compounds better than C18 or aqueous C18 phases under waterrich mobile phases. Under this condition, small changes in the acetonitrile fraction resulted in a marked increase in the retention of some polar drugs on the Si(PEO) phase. A typical condition observed in per aqueous liquid chromatography separations, a more environmentally friendly liquid chromatography approach. On the other hand, hydrophobic compounds showed lower mass transfer rates due to their low solubility in the aqueous mobile phase. Thus, the Si(PEO) phase was more suitable and efficient for separating polar or hydrophilic compounds.

Preparation and application of Au nanoparticles-decorated SO3H-cofunctionalized silica stationary phase for per aqueous liquid chromatography

Au nanoparticles (Au NPs)-decorated SO3H-cofunctionalized porous silica particles was prepared as a novel mixed-mode stationary phase and used in Per aqueous liquid chromatography (PALC) mode. The obtained stationary phase was characterized by elemental analysis, FT-IR spectra, and transmission electron microscopy. To evaluate the retention behavior, the effect of a variety of chromatographic factors on the retention of different types of solutes was investigated. The obtained column exhibited a hydrophobic interaction/cation-exchange mixed-mode retention mechanism, and a typical PALC characteristic was observed in water-rich mobile phases. When the particle size of Au NPs was 10–20 nm, the analytes had a sharp peak shape and the column pressure was also moderate. Compared with the hydrophilic interaction liquid chromatography (HILIC) and C18 column, some hydrophilic compounds such as six monosaccharides were baseline separated with the enhanced resolution of the obtained column under the PALC mode. Moreover, the efficiency of the Au-S/SO3H-SiO2-2 column was significantly higher than that of the HILIC column. PALC mode was a green annlysis method and suitable for the replacement of hydrophilic interaction liquid chromatography.

Polar polymer-immobilized stationary phase for aqueous reversed-phase liquid chromatography

In this work, a polar stationary phase was prepared by thermal immobilization of poly(glycidoxypropylmethyl-co-dimethylsiloxane) onto silica particles to separate polar compounds using water-rich mobile phases. The retention properties of the stationary phase were characterized by the separation of Tanaka and NIST SRM® 870 test mixtures. The elution order of the compounds indicated the reversed-phase mode, but the stationary phase was less hydrophobic and with higher silanophilic activity than typical stationary phases. Polar compounds presented reasonable retention in the stationary phase and symmetrical peaks in the chromatograms, due to hydrophilic interactions with glycidoxy groups in the polymer side chain. The stationary phase was applied for the separation of small polar and ionizable compounds. The retention factors increased with the aqueous mobile phase component. So, all polar compounds were separated efficiently and eluted symmetrically under highly aqueous mobile phases, >90%. Per aqueous liquid chromatography was the principal separation mode of the new stationary phase, presenting as a green alternative to the hydrophilic interaction liquid chromatography mode for the separation of polar compounds.

Analysis of gentisic acid and related renal cell carcinoma biomarkers using reversed-phase liquid chromatography with water-rich mobile phases

The problem of longer retention times using water-rich mobile phases in reversed-phase liquid chromatography (RPLC) has been addressed using hydrophobic alcohols such as butanol in very low quantities (approximately 0.1%) as the organic modifier. Advantages of water-rich mobile phases in RPLC for the separation of water-soluble and weakly retained compounds are improved separation of congeners and better tuning of RPLC separations. This is demonstrated in the separation of gentisic acid and related renal cell carcinoma (RCC) biomarkers in urine with a Zorbax C18 column and a mobile phase of 0.1% (volume/volume) butanol in water with 0.6% (volume/volume) acetic acid. Calibration curves for the RCC biomarkers were linear over the concentration range investigated (5–1000 ppm). Detection limits for the RCC biomarkers were 0.85 ppm (quinolinic acid), 1.75 ppm (gentisic acid), and 1.25 ppm (4-hydroxybenzoic acid). Recovery tests using synthetic urine samples containing 20 ppm, 100 ppm, and 700 pm of each RCC biomarker were successful for all compounds.

Investigations into the interaction thermodynamics of TRAP-related peptides with a temperature-responsive polymer-bonded porous silica stationary phase

The interaction thermodynamics of the thrombin receptor agonistic peptide (TRAP-1), H-Ser-Phe-Leu-Leu-Arg-Asn-Pro-OH, and a set of alanine scan substitution peptides, have been investigated with an n-octadecylacrylic polymer-bonded porous silica (Sil-ODA18) and water-acetonitrile mobile phases at temperatures ranging from 5 to 80 °C in 5 °C increments. The retention of these peptides on the Sil-ODA18 stationary phase decreased as the water content in the mobile phase was lowered from 80% (v/v) to ca. 45% (v/v) and reached a minimum value for each peptide at a specific water-acetonitrile composition. Further decreases in the water content of the mobile phase led to increased retention. The magnitude of the changes in enthalpy of interaction, ΔHassoc0, changes in entropy of interaction, ΔSassoc0, and changes in heat capacity, ΔCp0, were found to be dependent on the molecular properties of the mobile phase, the temperature, the structure/mobility of the stationary phase, and the conformation and solvation state of the peptides. With water-rich mobile phases, the retention behaviour of the TRAP analogues was dominated by enthalpic processes, consistent with the participation of strong hydrogen bonding effects, but became dominated by entropic effects with acetonitrile-rich mobile phases as the temperature was increased. These changes in the retention behaviour of these TRAP peptides are consistent with the generation of water or acetonitrile clusters in the mobile phase depending on the volume fractions of the organic solvent as the Sil-ODA18 stationary phase transitions from its crystalline to its isotropic state.

Mobile phase effects in reversed-phase and hydrophilic interaction liquid chromatography revisited

Correct adjustment of the mobile phase is equally important as the selection of the appropriate column for the separation of polar compounds in LC. Both solvophobic and selective polar interactions control the retention in the Reversed Phase and Hydrophilic Interaction modes. The retention models describing the effects of the volume fraction of the strong eluent component in binary mobile phases on the sample retention factors apply in a limited mobile phase composition range. We introduced a three-parameter retention model, which provides improved prediction of retention over a broad mobile phase range, under isocratic and gradient elution conditions. The model does not imply any assumptions concerning either adsorption or partition distribution mechanism, but allows estimating retention in pure strong and in pure weak mobile phase components. The experimental retention data for phenolic acids and flavones on several core-shell columns with different types of stationary phases agree with the theory.Many polar columns with important structural hydrophobic moieties show dual retention mechanism, (Reversed Phase in water rich mobile phases and Hydrophilic Interaction at high acetonitrile concentrations). It is possible to select the mobile phase compositions in each of the two modes for separations of samples containing compounds largely differing in polarity. The three-parameter model describes the retention in each mode, with separately determined best-fit parameters. We applied the two-mode model to the retention data of sulfonamides and benzoic acid related compounds on a new polymethacrylate zwitterionic monolithic micro-column.

1H-Tetrazole-5-amine Immobilized on Substituted Polymer Gel/Silica as a New Stationary Phase for Hydrophilic Interaction Chromatography

1H-Tetrazole-5-amine immobilized on substituted polymer gel/silica as a new stationary phase has been newly suggested for application in hydrophilic liquid chromatography (HILIC). The newly investigated column demonstrated a satisfactory retention of very polar compounds. The column showed mixed separation modes: reversed-phase (RP) in water-rich mobile phases and HILIC at high concentrations of acetonitrile content (> 60–80%) in aqueous–organic mobile phases. A continuous change in retention was observed at decreasing concentration of acetonitrile in water, the rise resulting in characteristic U-shape plots of retention factors versus the concentration of acetonitrile in the mobile phase, with minima corresponding to the transition between the mechanisms controlling the retention. The retention mechanism of the new column was investigated by the models (partitioning and surface adsorption) through adjustment of the water/acetonitrile ratio in the mobile phase, and by the influence of salt concentration, buffer pH, and temperature on the retention of tested analytes. The retention behavior of tested compounds on three different stationary phases (Atlantis HILIC—silica phase; TSKgel Amide-80—amide phase; Kromasil 60-5 DIOL—diol phase) was investigated to compare the selectivity with the new DCpak PTZ HILIC prototype column. The applicability of the new HILIC column for the chromatography of polar compounds was proven by the separation of mixtures of nucleobases (uracil, thymine, guanine, cytosine, and adenine) and organic acids (ascorbic, isoascorbic, pantothenic, and nicotinic acid). The new DCpak PTZ HILIC column could be a good alternative for the separation of the polar compounds.

Development and validation of high performance liquid chromatographic method for determination of gentisic acid and related Renal Cell Carcinoma biomarkers in urine

A reversed phase liquid chromatographic (RPLC) method was developed to simultaneously detect and quantify creatinine, quinolinic acid, gentisic acid and 4-hydroxybenzoic acid in urine. These four bio-markers are present in relatively high concentrations in urine. Using a 5% methanol in water mobile phase with 0.6% acetic acid and a Zorbax C18 column, baseline resolution for all four biomarkers in synthetic urine was achieved. Better resolution was obtained for the separation of these four compounds when water rich mobile phases were used. Detection of the four biomarkers in urine using the proposed RPLC method is limited by background from the urine matrix for the later eluting compounds and from the dead marker for earlier eluting compounds.

The synthesis of Gemini-type sulfobetaine based hybrid monolith and its application in hydrophilic interaction chromatography for small polar molecular

In this work, a novel Gemini-type sulfobetaine-based hybrid monolith was fabricated by co-polymerization of a homemade Gemini-type sufobetaine, 1,3-bis (N-(3-sulfopropyl)-N-(methacrylic acid-2-hydroxy propyl ester)-N-methyl ammonium)-propane (BSMMP) with vinyltrimethoxysilane (VTMS) and tetramethoxysilane (TMOS) in the presence of 2′-azobis (2-methylpropionamidine) dihydrochloride (V50) as initiator. The recipe of pre-copolymerization solution for monolith preparation was optimized carefully, and the resulting monolith was characterized by scan electron microscope (SEM), fourier transform infrared spectroscopy (FT-IR) and thermal gravimetry analysis (TGA). The results indicated that the optimized monolith possess homogeneous bio-continue pore structure, and a relatively high proportion of the sulfobetaine groups was successfully introduced into the monolith. The results from permeability test shows that the proposed monoliths have excellent permeability within both water rich mobile phase and acetonitrile rich mobile phase, due to the rigid inorganic framework of hybrid monolith. Results showed that the relative standard deviations (RSDs) of run-to-run, column-to-column and batch-to-batch were less than 1.9%, 3.7% and 6.2%, respectively. The lowest plate height value of the column for thiourea was less than 10µm. Furthermore, separating the polar small molecules including nucleosides, the base species, the phenols and the amides were performed within 14min, 22min 18min and 9min on the prepared monolith, respectively, good separation effect on these polar small molecules was found, and remarkable hydrophilic retention behavior was observed when an acetonitrile rich mobile phase was applied, attributing to the existence of strong polar Gemini-type sulfobetaine groups. Finally, the potential application of the resulting monolith in complex sample was successfully demonstrated by separating a BSA digest in gradient elution mode.

Modified QuEChERS as a novel sample preparation method for analysis of N-nitrosodiethanolamine in shampoo by high performance liquid chromatography

This study sought to develop a novel method for the trace analysis ofN-nitrosodiethanolamine (NDELA) in shampoos inspired by a Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) extraction, followed by an RP-HPLC method with a water-rich mobile phase.

Dual Retention Mechanism in Two-Dimensional LC Separations of Barbiturates, Sulfonamides, Nucleic Bases and Nucleosides on Polymethacrylate Zwitterionic Monolithic Micro-Columns

In-house prepared zwitterionic polymethacrylate micro-columns using in situ polymerization of N,N-dimethyl-N-metacryloxyethyl-N-(3-sulfopropyl) ammonium betaine (MEDSA) functional monomer with bisphenol A glycerolate dimethacrylate (BIGDMA) cross-linker provided excellent stability and reproducibility of preparation and separation efficiency of 60,000–70,000 theoretical plates m−1 for small molecules under isocratic conditions. The column showed a dual retention mechanism, reversed-phase (RP) in highly aqueous mobile phases and aqueous normal-phase (HILIC) in acetonitrile-rich mobile phases. This property can be used to obtain complementary separation and combined information on the sample from repeated injections of a sample on a single column, in different mobile phases characteristic for the HILIC and for the RP modes, which is in fact a form of offline two-dimensional chromatography on a single column. The dual retention mechanism has been observed with a variety of columns, however, often with impractically narrow retention range in one of the two modes. To take full advantage from the combined single-column RP–HILIC experiments, the column should provide a sufficiently broad mobile phase interval both in the RP and in the HILIC mode. The BIGDMA-MEDSA micro-columns proved suitable earlier for the combined RP–HILIC separations of some phenolic compounds and flavonoids. In the present work, we investigated the effects of the mobile phase composition on the retention of a variety of polar compounds over full retention range of buffered aqueous acetonitrile mobile phases, to find potentially useful HILIC and RP retention ranges for barbiturates, sulfonamides, nucleosides and nucleic bases. In the HILIC mode, proton donor–acceptor interactions show a major effect on retention and selectivity of separation, whereas the size of the non-polar hydrocarbon part of the sample molecule is the most important factor in the water-rich mobile phases. The sample structure strongly affects the composition of aqueous–organic mobile phases at which the transition between the two retention modes occurs. Of the investigated sample types, barbiturates show better separation under reversed-phase conditions, whereas nucleosides and nucleic bases in the HILIC mode. Aromatic carboxylic acids and sulfonamides can be separated either in the reversed phase or under HILIC conditions, the two separation modes showing complementary selectivity of separation.

Histidine-modified organic-silica hybrid monolithic column for mixed-mode per aqueous and ion-exchange capillary electrochromatography.

A novel organic-silica hybrid monolith was prepared through the binding of histidine onto the surface of monolithic matrix for mixed-mode per aqueous and ion-exchange capillary electrochromatography. The imidazolium and amino groups on the surface of the monolithic stationary phase were used to generate an anodic electro-osmotic flow as well as to provide electrostatic interaction sites for the charged compounds at low pH. Typical per aqueous chromatographic behavior was observed in water-rich mobile phases. Various polar and hydrophilic analytes were selected to evaluate the characteristics and chromatographic performance of the obtained monolith. Under per aqueous conditions, the mixed-mode mechanism of hydrophobic and ion-exchange interactions was observed and the resultant monolithic column proved to be very versatile for the efficient separations of these polar and hydrophilic compounds (including amides, nucleosides and nucleotide bases, benzoic acid derivatives, and amino acids) in highly aqueous mobile phases. The successful applications suggested that the histidine-modified organic-silica hybrid monolithic column could offer a wide range of retention behaviors and flexible selectivities toward polar and hydrophilic compounds.

A novel imidazolium-based organic–silica hybrid monolith for per aqueous capillary electrochromatography

A new imidazolium-based organic–silica hybrid monolith by binding the imidazole-1-acetic acid onto the surface of monolithic silica was prepared through a simple route. The obtained monolith exhibited per aqueous capillary electrochromatography (PACEC) characteristics in water-rich mobile phases. A typical per aqueous chromatography behavior was observed for the test solutes (an increase in H2O content resulted in an increase in retention). The effects of water content, pH and ion strength of mobile phase on the retention of test compounds in highly aqueous eluents were investigated in detail. PACEC in itself is a green chromatography mode that makes green capillary electrochromatography possible. Various hydrophilic compounds including amino acids were baseline separated with enhanced resolution of the obtained monolith under PACEC mode. It is evidenced that solutes were retained predominantly by hydrophobic interactions under PACEC conditions. Therefore, PACEC has the potential to replace hydrophilic interaction chromatography and meanwhile it is complementary to the reverse-phase chromatography.

Effect of the water content on the retention and enantioselectivity of albendazole and fenbendazole sulfoxides using amylose-based chiral stationary phases in organic–aqueous conditions

Four commercially available immobilized amylose-derived CSPs (Chiralpak IA-3, Chiralpak ID-3, Chiralpak IE-3 and Chiralpak IF-3) were used in the HPLC analysis of the chiral sulfoxides albendazole (ABZ-SO) and fenbendazole (FBZ-SO) and their in vivo sulfide precursor (ABZ and FBZ) and sulfone metabolite (ABZ-SO2 and FBZ-SO2) under organic–aqueous mode. U-shape retention maps, established by varying the water content in the acetonitrile- and ethanol–water mobile phases, were indicative of two retention mechanisms operating on the same CSP. The dual retention behavior of polysaccharide-based CSPs was exploited to design greener enantioselective and chemoselective separations in a short time frame. The enantiomers of ABZ-SO and FBZ-SO were baseline resolved with water-rich mobile phases (with the main component usually being 50–65% water in acetonitrile) on the IF-3 CSP and ethanol–water 100:5 mixture on the IA-3 and IE-3 CSPs. A simultaneous separation of ABZ (or FBZ), enantiomers of the corresponding sulfoxide and sulfone was achieved on the IA-3 using ethanol–water 100:60 (acetonitrile–water 100:100 for FBZ) as a mobile phase.

Hydrophilic interaction liquid chromatography columns classification by effect of solvation and chemometric methods

In the current work, a 14 different types of stationary phases with specific structural properties (eight commercially available stationary phases and six home-made) have been studied. We used the minor disturbance method to measure the excess adsorption isotherms of water onto surface of chemically bonded stationary phases from water–acetonitrile mixtures. The presence of polar and hydrophobic groups in the structures of adsorbents as well as changes in the mobile phase composition causes the excess adsorption of given solvent when its concentration in the mobile phase is low. The excess adsorption of water is observed in acetonitrile-rich mobile phase and the excess adsorption of acetonitrile is observed in water-rich mobile phase. The maximum excess of adsorbed water is connected with a negative excess of adsorbed acetonitrile. However, the scale of these excess adsorption depend on the type of the stationary bonded phases. The retention factors of three test solutes (tryptophan, glycine and proline) are correlated with the maximum amount of excessively adsorbed water on the stationary bonded phase surface. Linear dependence of retention factor with excess amount of water suggest, that the amount of adsorbed water (“hydrophilic pillow”) play an important role in the retention mechanism in HILIC. All tested stationary phases were divided into several groups according to the retention factors of 16 different biologically active compounds (selected amino acids, pesticides, vitamin B6). Principal component analysis (PCA) and cluster analysis (CA) analysis were used in column comparison and grouping. CA results indicated that all stationary phases may be generally grouped into several clusters, due to structure and properties of stationary phases. Interesting results were obtained also with the use of PCA. Presented methodologycan provide useful information on the hydrophilic properties of various polar columns and their suitability for HILIC applications.

Introducing Enantioselective Ultrahigh-Pressure Liquid Chromatography (eUHPLC): Theoretical Inspections and Ultrafast Separations on a New Sub-2-μm Whelk-O1 Stationary Phase

A new chiral stationary phase for ultrahigh-pressure liquid chromatography (UHPLC) applications was prepared by covalent attachment of the Whelk-O1 selector to spherical, high-surface-area 1.7-μm porous silica particles. Columns of varying dimensions (lengths of 50, 75, 100, and 150 mm and internal diameters of 3.0 or 4.6 mm) were packed and characterized in terms of permeability, efficiency, retention, and enantioselectivity, using both organic and water-rich mobile phases. A conventional HPLC Whelk-O1 column based on 5.0-μm porous silica particles and packed in a 250 mm × 4.6 mm column was used as a reference. Van Deemter curves, generated with low-molecular-weight solutes on a 100 mm × 4.6 mm column packed with the 1.7-μm particles, showed H(min) (μm) and μ(opt) (mm/s) values of 4.10 and 5.22 under normal-phase and 3.74 and 4.34 under reversed-phase elution conditions. The flat C term of the van Deemter curves observed with the 1.7-μm particles allowed the use of higher-than-optimal flow rates without significant efficiency loss. Kinetic plots constructed from van Deemter data confirmed the ability of the column packed with the 1.7-μm particles to afford subminute separations with good efficiency and its superior performances in the high-speed regime, compared to the column packed with 5.0-μm particles. Resolutions in the time scale of seconds were obtained using a 50-mm-long column in the normal phase or polar organic mode. The intrinsic kinetic performances of 1.7-μm silica particles are retained in the Whelk-O1 chiral stationary phase, clearly demonstrating the potentials of enantioselective UHPLC in terms of high speed, throughput, and resolution.

Analysis of vanilla extract by reversed phase liquid chromatography using water rich mobile phases

Vanillin is the major constituent of vanilla extract, a flavoring ingredient used in food and beverages. Natural vanilla extract prepared from the bean of the tropical orchid, Vanilla planifolia, is expensive due to the limited supply of the vanilla bean. For this reason, synthetic vanilla extracts are widely used. Synthetic vanilla extracts are less complex and usually contain vanillin, ethyl vanillin, and other related compounds that are prepared from inexpensive starting materials. Several liquid chromatographic methods have been developed to quantitate coumarin, vanillin, and ethyl vanillin in vanilla extract. The use of water rich mobile phases in reversed phase liquid chromatography (RPLC), e.g., 1% butanol in water with 0.2% acetic acid with C18, C8, and cyanopropyl columns, has been investigated as a potential method to characterize the composition of synthetic vanilla extracts. Better resolution is achieved in the separation of vanillin compounds when hydrophobic alcohols are used as organic modifiers. This can be attributed to butanol partitioning into the bonded phase, which provides a more extended ordered surface increasing the contact surface area of the stationary phase and thereby increasing the selectivity of the separation. Using water rich mobile phases, constituents of vanilla extract in 36 commercial products obtained from stores in the local area were identified demonstrating the efficacy of the proposed RPLC method.

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.

LC-PDA-MS Studies of the Photochemical Degradation of Imidacloprid

A liquid chromatographic (LC) method that uses a water rich mobile phase to isolate imidacloprid and its photolysis products, a photodiode array (PDA), and electro-spray mass spectrometer (MS) to detect them was employed to characterize mixtures of imidacloprid and its degradation products. The photolysis study that was undertaken showed that decomposition of imidacloprid in distilled water is a first order reaction, and the major photolysis products are imidacloprid urea, imidacloprid olefin, and imidacloprid desnitro.

Preparation and characterization of polymethacrylate monolithic capillary columns with dual hydrophilic interaction reversed‐phase retention mechanism for polar compounds

Monolithic columns for capillary hydrophilic interaction liquid chromatography (HILIC) were prepared in fused-silica capillaries by radical co-polymerization of [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide and ethylene dimethacrylate in various binary and ternary porogen solvent mixtures with azobisisobutyronitrile as the initiator of the polymerization reaction. Columns showed mixed separation modes: reversed-phase (RP) in water-rich mobile phases and HILIC at high concentrations of acetonitrile (>60-80%) in aqueous-organic mobile phases. A continuous change in retention was observed at increasing concentration of water in acetonitrile, giving rise to characteristic U-turn plots of retention factors versus the concentration of water in the mobile phase, with minima corresponding to the transition between the mechanisms controlling the retention. The selectivity of organic polymer monolithic columns for HILIC separations can be varied by adjusting the concentration of sulfobetaine monomer and the composition of the porogen solvent in the polymerization mixture. Under HILIC conditions, the monolithic capillary sulfobetaine columns show separation selectivities for polar phenolic acids similar to those of a commercial silica-based sulfobetaine ZIC-HILIC column, which, however, has limited selectivity in the RP mode due to lower retention.