Stationary Phase For High-performance Liquid Chromatography Research Articles

Synthesis of monolithic HPLC stationary phase with self‐assembled molecular recognition sites for 4‐aminophenol

AbstractA molecularly imprinted polymer (MIP) monolith for selective recognition of 4‐aminophenol (4‐AMP) was prepared by in situ polymerization technique as high‐performance liquid chromatography (HPLC) stationary phase. For this purpose, several 4‐AMP imprinted monoliths were synthesized by using only methacrylic acid (MAA), acrylamide (AAM), or isobornyl methacrylate (IBMA) in the presence of high amount of crosslinker, ethylene glycol dimethacrylate (EDMA), and these polymeric monolith columns were connected to HPLC to evaluate their separation capabilities. By selection of appropriate functional monomer and optimization of polymerization conditions, MAA‐based monolithic MIP showed good flow through properties, high selectivity to the templated molecule, and high resolution in the separation of paracetamol and its main impurity, 4‐AMP. Besides, effective binding site density and dissociation constant of this monolith were estimated by using frontal chromatography and found as 7.95 μmol/g and 1.06 mM, respectively. Surface area of the same monolith was found as 23.48 m2/g from multipoint BET analysis. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011

Synthesis and Chiral Recognition Ability of a Poly(phenylenevinylene)-Encapsulated Amylose Derivative

Abstract Poly(p-phenylenevinylene) (PPV) was found to be encapsulated in amylose during the polymerization of the precursor monomer in an aqueous solution. The resulting amylose–PPV composite can be further chemically modified by introducing various substituents into the hydroxy groups of the amylose using isocyanates and acetic anhydride. Here we report on the chemical modification of the amylose–PPV composite with 3,5-dimethylphenyl isocyanate in order to obtain a novel chiral packing material for resolving enantiomers by high-performance liquid chromatography (HPLC). The obtained 3,5-dimethylphenylcarbamated amylose–PPV was soluble in pyridine and exhibited a lyotropic liquid crystalline phase in a concentrated pyridine solution. The chiral recognition ability of the 3,5-dimethylphenylcarbamated amylose–PPV was different from that of amylose tris(3,5-dimethylphenylcarbamate) having no PPV rods in the helical cavity of the amylose and can resolve many racemic compounds into enantiomers. Among racemic compounds, cyclic dibenzamide and dibenzanilide derivatives were resolved on the 3,5-dimethylphenylcarbamated amylose–PPV better than amylose tris(3,5-dimethylphenylcarbamate). The difference in the enantioseparation abilities of these amylose-based chiral stationary phases for HPLC is discussed based on the difference in their helical structures.

Evaluations of poly(methyl methacrylate) bearing glucose pendant or cellobiose pendant in high performance liquid chromatography as polymeric chiral stationary phases

The glucopyranos units consisting of cyclodextrin and polysaccharide derivatives have been extensively used as chromatographic chiral stationary phases. The synthetic polymeric chiral stationary phases based on methyl methacrylate bearing glucose or cellobiose pendants for high performance liquid chromatography (HPLC) were prepared by radical polymerization. Fifteen racemic chiral compounds including alcohols, amines, amides and ketones, were resolved on the two chiral stationary phases using hexane-isopropanol (90/10, v/v), hexane-isopropanol-triethylamine (90/10/0.2, v/v/v) or hexane-isopropanol-trifluoroacetic acid (90/ 10/0.2, v/v/v) as mobile phase, separately. The results showed that both chiral stationary phases possessed good enantioselectivity for most of the racemic alcohols and amines. A few amides and ketones could also be separated. There was some compensation of the chiral discriminating abilities between the two chiral stationary phases. This work indicates that polymers of mono- and disaccharide derivatives can soon become a very attractive new chiral stationary phase for HPLC.

Reversed‐phase HPLC of peptides: Assessing column and solvent selectivity on standard, polar‐embedded and polar endcapped columns

We desired to evaluate the chromatographic selectivity for peptides of silica-based RP high-performance liquid chromatography stationary phases with various modifications (polar embedding and polar endcapping on C(18) columns; ether-linked phenyl column with polar endcapping) compared with n-alkyl (C(18), C(8)) and aromatic phenylhexyl columns. Thus, we have designed and synthesized two series of synthetic peptide standards with the sequence Gly-Gly-Leu-Gly-Gly-Ala-Leu-Gly-X-Leu-Lys-Lys-amide, where the N-terminal either contains a free α-amino group (AmC series) or is N(α)-acetylated (AcC series) and where position X is substituted by Gly, Ala, Val, Ile, Phe or Tyr. These represent series of peptides with single substitutions of n-alkyl (Gly<Ala<Val<Ile in order of increasing hydrophobicity), alkyl versus aromatic side-chains (Phe, Tyr) and a side-chain also with polar character (Tyr). Peptide mixtures were run on all columns at pH 2 (linear gradients of ACN or methanol in aqueous mobile phases). Our initial results clearly demonstrate the useful complementarity of different RP high-performance liquid chromatography packing materials, with peptide mixtures showing profound selectivity differences between n-alkyl versus polar-embedded and polar endcapped C(18) stationary phases. In addition, changing the polarity of the RP matrix in combination with changing the polarity of the mobile phase (ACN versus methanol) is shown to be synergistic to maximize selectivity of peptide separations.

Preparation and Evaluation of Silica-Based Ionic Liquid-Modified Stationary Phase for HPLC

A new high-performance liquid chromatography (HPLC) stationary phase was firstly synthesized based on the ionic liquid 2-ethyl-4-methylimidazole chloride. And the performances of the resulting stationary phase were determined by Fourier transform infrared (FT-IR), thermogravimetric analysis (TGA), and (13)C nuclear magnetic resonance (NMR) spectroscopy. The retention characteristics of the resulting stationary phase were evaluated by normal phase HPLC conditions to separate tanshinone I and tanshinone IIA in Danshen herb. The results demonstrated that the synthesized ionic liquid was bond-linked to the silica surface successfully and that IL-modified silica is more effective than unmodified silica when separating tanshinone I and tanshinone IIA in Danshen herb.

Silica–MOF Composites as a Stationary Phase in Liquid Chromatography

AbstractMetal‐organic framework materials (MOFs) are a class of microporous and crystalline materials with great potential for adsorption‐based separations. Harnessing the separation ability of these materials in high‐performance liquid chromatography (HPLC) requires the use of small and uniform particles in order to achieve good column packing. The well‐known MOF material [Cu3(BTC)2] is, however, typically synthesized as a polydisperse mixture due to its nucleation and growth mechanism. It is demonstrated how a new synthesis method for [Cu3(BTC)2] enables the formation of the MOF inside the pores of silica beads often used in chromatography, leading to monodisperse silica–MOF composite spheres with a uniform particle size of 3 μm. When employed as an HPLC stationary phase, this material combines the good column packing properties of the silica and separation ability of the MOF material.

Application and development of monolithic stationary phases for HPLC in pharmaceutical analysis and separation

In recent years, monolithic supports as stationary phases in high-performance liquid chromatography (HPLC) have become a rapidly burgeoning field. This review summarizes the current achievements and their application in pharmaceutical analysis and separation. One kind is the silica monoliths prepared from tetraalkoxysilane by a sol-gel method with either micrometer-size through-pores or high specific surface areas and well suited for small molecules in HPLC modes. The other kind is the organic polymer-based monoliths including monolithic molecularly imprinted polymers in treatments of the samples before injected into the chromatograph in biological fluids. The analytical potential of these columns is demonstrated with separations involving various drugs in different chromatographic modes.

TYPE C AMINO COLUMNS FOR AFFINITY AND AQUEOUS NORMAL PHASE CHROMATOGRAPHY: SYNTHESIS AND HPLC EVALUATION

The synthesis of novel stationary phases for the retention of polar compounds and for affinity chromatography was presented. Two amino compounds, 4-diethylamino-2-butyn-1-ol and 3-amino-3-methyl-1-butyne, were bonded to a silica hydride surface using Silica-C and a hydrosilation procedure. The bonding was confirmed using CP – MAS NMR and FTIR techniques. High Performance Liquid Chromatography (HPLC) evaluation of the synthesized HPLC stationary phases was performed using the Neue Test. Aqueous Normal Phase (ANP) properties of synthesized amino phases were confirmed using a broad range of polar compounds. Type III-O trypsin inhibitor from chicken egg white was used to test the affinity aspect of the amino bonded trypsin column.

Surfactant-bound monolithic columns for separation of proteins in capillary high performance liquid chromatography

A surfactant-bound monolithic stationary phase based on the co-polymerization of 11-acrylamino-undecanoic acid (AAUA) is designed for capillary high performance liquid chromatography (HPLC). Using D-optimal design, the effect of the polymerization mixture (concentrations of monomer, crosslinker and porogens) on the chromatographic performance (resolution and analysis time) of the AAUA–EDMA monolithic column was evaluated. The polymerization mixture was optimized using three proteins as model test solutes. The D-optimal design indicates a strong dependence of chromatographic parameters on the concentration of porogens (1,4-butanediol and water) in the polymerization mixture. Optimized solutions for fast separation and high resolution separation, respectively, were obtained using the proposed multivariate optimization. Differences less than 6.8% between the predicted and the experimental values in terms of resolution and retention time indeed confirmed that the proposed approach is practical. Using the optimized column, fast separation of proteins could be obtained in 2.5 min, and a tryptic digest of myoglobin was successfully separated on the high resolution column. The physical properties ( i.e., morphology, porosity and permeability) of the optimized monolithic column were thoroughly investigated. It appears that this surfactant-bound monolith may have a great potential as a new generation of capillary HPLC stationary phase.

Accelerating the Quality Control of Pharmaceuticals Using Monolithic Stationary Phases: A Review of Recent HPLC Applications

High-performance liquid chromatography (HPLC) is the predominant technique in the quality control of pharmaceutical formulations. HPLC offers highly sensitive and selective analytical methods with increased robustness and separation efficiency. The majority of HPLC-based methodologies are based on the usage of particulate-based columns and cannot be applied at elevated flow rates (> 2 mL/min) due to excessive back-pressure. Therefore, the typical duration of separation cycles is in the range of 5-30 min, making most assays time-consuming, especially when a lot of samples have to be analyzed in a minimum amount of time. Monolithic stationary phases for HPLC offer a very interesting alternative to conventional particulate-based columns. The size of the skeleton and the distribution of the pores of monolithic materials offer the possibility of developing efficient separation protocols at higher flow-rates due to the low pressure-drop across the column. The present review intends to cover the applications of monolithic based HPLC stationary phases in the quality control of pharmaceutical formulations, including identification of active pharmaceutical ingredient, assay, purity, dissolution, blending, and dosage uniformity, etc.

Separation and Quantitative Analysis of Alkyl Sulfate Ethoxymers by HPLC

Separation of alkyl sulfate ethoxymers is investigated on various high-performance liquid chromatography (HPLC) stationary phases: Acclaim C18 Surfactant, Surfactant C8, and Hypercarb. For a fixed alkyl chain length, ethoxymers are eluted in the order of increasing number of ethoxylated units on Acclaim C18 Surfactant, whereas a reversed elution order is observed on Surfactant C8 and Hypercarb. Moreover, on an Acclaim C18 Surfactant column, non-ethoxylated compounds are eluted in their ethoxymers distribution and the use of sodium acetate additive in mobile phase leads to a co-elution of ethoxymers. HPLC stationary phases dedicated to surfactants analysis are evaluated by means of the Tanaka test. Surfactant C8 presents a great silanol activity whereas Acclaim C18 Surfactant shows a high steric selectivity. For alkyl sulfates, linearity of the calibration curve and limits of detection and quantitation are evaluated. The amount of sodium laureth sulfate raw material found in commercial body product is in agreement with the specification of the manufacturer.

Stereospecificity of Daidzein metabolism leading to S-Equol by human intestinal bacterium

S-Equol, a metabolite of the isoflavonoid daidzein in the human intestine, shows the most active estrogen activity. Stereospecific production of S-equol by a human intestinal anaerobic bacterium strain Julong732 was studied. Julong732 was known to metabolize dihydrodaidzein (DHD) into S-equol tetrahydrodaidzein (THD) [1] but its metabolic stereospecificity has not yet been elucidated. DHD and THD were prepared, and their stereoisomers were separated by reverse-phase and chiral stationary-phase high-performance liquid chromatography. The absolute configurations of DHD enantiomers and four THD stereoisomers were assigned by 1H NMR, 13C NMR, and CD spectroscopies [2,3]. The reaction of the cell-free extract with DHD showed that only 3S-DHD was transformed into 3S-equol. When each of the four stereoisomers of THD was reacted with the cell-free extract of Julong732, only 3R,4S-THD was metabolized by the extract, producing S-equol. 7,4'-Dihydroxyisoflav-3-ene (dehydroequol, DE), a putative intermediate during the anaerobic metabolism of DHD, was unable to produce S-equol.

Polyethylene glycol diacrylate-based supermacroporous monolithic cryogel as high-performance liquid chromatography stationary phase for protein and polymeric nanoparticle separation

A supermacroporous monolithic cryogel was directly prepared by in situ cryo-copolymerization in a stainless steel cartridge (70 mm × 5.0 mm I.D.) using methacrylic acid (MAA) as functional monomer and polyethylene glycol diacrylate (PEGDA) as crosslinker. The highly crosslinked (90%, molar ratio) poly(MAA–PEGDA) cryogel had more uniform supermacropores with a mean diameter of 25 μm compared to the poly(acrylamide)-based cryogels. The viability of poly(MAA–PEGDA) cryogel as a medium was demonstrated for separations of lysozyme from chicken egg white (CEW) and water-soluble poly( N-isopropylacrylamide-co-3-(dimethylamino) propyl methacrylamide) (NIPAM-DMAPMA) nanoparticles from its crude reaction solution. The dynamic binding capacities of lysozyme and the polymeric nanoparticles were 4.51 × 10 −3 μmol/ml and 33.4 μg/ml, respectively. The lysozyme recovered from the above separations had a purity of more than 85%, and retained 90% of its enzymatic activity.

Novel reversed-phase high-performance liquid chromatography stationary phase with oligo(ethylene glycol) “click” to silica

Oligo(ethylene glycol) (OEG) covalently bonded silica was prepared by using click chemistry and employed as a stationary phase for reversed-phase high-performance liquid chromatography. The column efficiency and effect of organic modifier content on retention were investigated. The separation selectivity was also studied with phenyl compounds and an actual sample of natural products. The results indicated that the stationary phase possessed good separation efficiency and separation selectivity in RP-HPLC mode. Moreover, the stationary phase showed good complementary separation selectivity to the C18 stationary phase. The OEG stationary phase had “clustering” function for “homologous component” in the separation of natural products.

Comparative evaluation of high-performance liquid chromatography stationary phases used for the separation of peptides in terms of quantitative structure–retention relationships

Chromatographic measurements were made on 17 physicochemically diversified high-performance liquid chromatography (HPLC) columns which were further analyzed in terms of their similarities and dissimilarities for 25 carefully designed, structurally diverse peptides showing distinctly distinguished groups. The goal of the study was to investigate the molecular mechanism of retention and to find an objective manner of quantitative comparison of retention properties and classification of modern stationary phases for reverse phase HPLC (RP-HPLC). We utilized the structural descriptors of peptides obtained from molecular modeling to describe their chromatographic retention behavior under given HPLC conditions. Quantitative structure–retention relationships (QSRR) with the following descriptors were employed: logarithm of the sum of amino acid retention contributions in a given peptide, log Sum AA, logarithm of the Van der Waals volume of the peptide, log VDW vol, and logarithm of its calculated n-octanol/water coefficient, clog P. The best QSRR equations were obtained in the case of monolithic and regular octadecylsilica columns. On the other hand, the combination of QSRR and principal component analysis (PCA) can be considered as the efficient tool allowing column classification and searching for orthogonal HPLC conditions required to separate peptides.

Preparation of normal‐phase HPLC stationary phase based on monodisperse hydrophilic polymeric beads and their application

AbstractThe monodisperse, 5.0 μm hydrophilic macroporous poly(glycidymethacrylate‐co‐ethylenedimethacrylate) beads were first prepared based on monosized linear poly(glycidylmethacrylate) beads as seed by using a single‐step swelling and polymerization method. The seed beads prepared by dispersion polymerization exhibited good absorption of the monomer phase. The pore size distribution of the beads was evaluated by mercury instrusion method. The surface area was calculated from the BET isotherms of nitrogen adsorption and desorption. The beads were modified to be a normal‐phase liquid chromatographic (NPLC) stationary phase for high performance liquid chromatography (HPLC) in the following steps. First, the beads were completely hydrolyzed. Second, hydrolyzed particles were reacted with epichlorihydrin followed by another hydrolysis of the newly introduced epoxide groups. The retention properties of the NPLC stationary phase were easily modulated by changes in the composition of the mobile phase. The performance of theses beads was demonstrated with the separation of a variety of polar compounds. The satisfactory results were obtained. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 2007

Asymmetric Polymerization of (S)-N-Maleoyl-L-Leucine Allyl Ester and Chiral Recognition Ability of Its Polymer as Chiral Stationary Phase for HPLC

(S)-N-Maleoyl-L-leucine allyl ester ((S)-ALMI), a kind of N-substituted maleimide having two double bonds with different reactivities, was newly synthesized from maleic anhydride, L-leucine, and allyl alcohol. Asymmetric polymerizations were carried out to obtain optically active polymers. Poly((S)-ALMI) obtained by anionic polymerization with n-butyllithium/(S,S)-(1-ethylpropylidene)-bis(4-benzyl-2-oxazoline) (n-BuLi/Bnbox) complex in THF showed the highest negative specific rotation of −429.1°. Chemoselective polymerization behaviors of (S)-ALMI on the two reactive groups were elucidated. In anionic polymerizations, only the double bond in the maleimide moiety exclusively took part in the polymerization, affording the corresponding poly((S)-ALMI) with the allyl pendant group. In radical polymerizations, the solvents strongly affect the radical polymerization behaviors of (S)-ALMI. Chiroptical properties and structures of the poly((S)-ALMI)s obtained were investigated by GPC, CD, XRD, and NMR measurements. The methods of preparing chiral stationary phases (CSPs) for high performance liquid chromatography (HPLC) and the chiral recognition ability of the optically active poly((S)-ALMI) were also discussed.

Mobile phase effects on retention on a new butylimidazolium-based high-performance liquid chromatographic stationary phase

A new HPLC stationary phase based on n-butylimidazolium bromide has been characterized by a linear solvation energy relationship (LSER) approach in the binary acetonitrile/water mobile phases. The retention properties of the stationary phase were systematically evaluated in terms of intermolecular interactions between 28 test solutes and the stationary phase. The results and further comparisons with conventional reversed phase system confirm that retention properties are similar to phenyl phases in acetonitrile/water mixtures. The results obtained with acetonitrile/water mixtures are also compared with results obtained using methanol/water mixtures.

Immobilized Polysaccharide-Based Chiral Stationary Phases for HPLC

Polysaccharide derivatives, such as phenylcarbamates and benzoates of cellulose and amylose, are known to show high chiral recognition abilities for many racemates when used as chiral stationary phases (CSPs) for high-performance liquid chromatography (HPLC). This type of CSPs has usually been prepared by coating the polysaccharide derivatives onto a macroporous silica gel without a chemical bond. Therefore, rather limited numbers of solvents can be used as eluents, and solvents, such as chloroform and tetrahydrofuran (THF), which swell or dissolve the polysaccharide derivatives cannot be used. The selection of a suitable eluent is very important for both analytical and preparative separations. In order to enhance the versatility of the polysaccharide derivative-based CSPs, the derivatives have to be immobilized. Here we review the immobilization methods of the polysaccharide derivatives mainly onto silica gel.

Direct enrichment and high performance liquid chromatography analysis of ultra-trace Bisphenol A in water samples with narrowly dispersible Bisphenol A imprinted polymeric microspheres column

Direct injection, enrichment and high performance liquid chromatography (HPLC) analysis of ultra-trace Bisphenol A (BPA) in water samples using one narrowly dispersible BPA imprinted polymeric microspheres (MIPM) column in one analysis process was developed. One BPA imprinted MIPM that had the best globular morphology and imprinted efficiency was used as HPLC stationary phase and applied to direct analysis of ultra-trace BPA in water. The optimum direct analysis conditions were: conditioning the MIPM column with water for 10 min, injecting 40 mL water sample directly, eluting with 70% methanol for 13 min and then 100% methanol for 7 min. Under the optimum conditions, the MIPM column can simultaneously extract, enrich, separate and determine ultra-trace BPA in one analysis process with UV detector by injection of large volume water samples (40 mL). The calibration graph was linear with R 2 > 0.998 in the ranges from 0.1–100 nmol/L BPA standard solution. The intra- and inter-day RSD are less than 9.5 and 9.6%, respectively. The limit of quantification was 0.1 nmol/L. RSD for spiked tap and lake water was less than 8.9% and the recoveries were 96–101.8%. The enrichment factor for BPA was 10,000 as 40 mL water sample was directly injected and analyzed.