Development of some stationary phases for reversed-phase high-performance liquid chromatography

The availability of a variety of stable organic stationary phases for columns has been a key factor in the development of HPLC as a major scientific tool. This paper explores the history and rationale used in the development of some important stationary phases and attempts to identify some of the strengths and limitations of these materials. Some of the author's experiences in stationary phase development illustrate approaches leading to present-day columns that exhibit a broad range of selectivity coupled with a high degree of reproducibility. Suggestions also are made for additional stationary phases that may be needed to complete column selectivity potential for HPLC separations.     

Chemically bonded multifunctional stationary phases for high-performance liquid chromatography

Summary A new class of stationary phases for high-performance liquid chromatography (HPLC) are described which simulate in their retention chracteristics ion-pair separations. The phases consist of mixtures of chemically dissimilar ligands chemically bonded to silica supports. These phases are largely reversed-phase in nature, but also contain significant ion-exchange properties, at levels similar to those demonstrated to occur in ionpairing. By bonding both ionic and hydrophobic groups in the correct proportions, mixed retention mechanisms are created, resulting in unique selectivities, while retaining the excellent stabilities and efficiencies characteristic of bonded phases. The ratio of hydrophobic to ionic character can be controlled during the synthesis, and is used as a tool to vary the stationary phase, rather than only the mobile phase, to effect the separation desired. The synthesis and behavior of both anionic and cationic/reversed-phase materials are described, and are applied to the simultaneous separation of nucleosides and nucleotides, and to the separation of the catecholamines.Presented at the 14th International Symposium on Chromatography London, September, 1982.     

Protein-based chiral stationary phases for high-performance liquid chromatography enantioseparations

The enantioseparations of various compounds using proteins as the chiral selectors in high-performance liquid chromatography (HPLC) are considered in this review. The proteins used include albumins such as bovine serum albumin and human serum albumin, glycoproteins such as alpha1-acid glycoprotein, ovomucoid, ovoglycoprotein, avidin and riboflavin binding protein, enzymes such as trypsin, alpha-chymotrypsin, cellobiohydrolase I, lysozyme, pepsin and amyloglucosidase, and other proteins such as ovotransferrin and beta-lactoglobulin. This review deals with the properties of HPLC chiral stationary phases based on proteins, and the enantioselective properties and chiral recognition mechanisms of these stationary phases.     

Imprinted chiral stationary phases in high-performance liquid chromatography

Polymers imprinted with chiral templates offer a new generation of tailor-made chiral stationary phases (CSPs) with predictable selectivities. This review summarizes the present state of the art of molecular imprinting to generate tailor-made CSPs and provides an overview of the main factors involved in the manufacturing process that are crucial to the chromatographic performance of the phases.     

Trends in stationary phases in high-performance liquid chromatography

There has been a major breakthrough in the design and synthesis of selective stationary phases in reversed-phase, ion-exchange, size-exclusion and affinity mode of high-performance liquid chromatography. Tailored stationary phases now have widespread applications in sample clean-up by solid phase extraction, as sorbents in microbore, analytical and preparative columns, and in large-scale separations.     

Synthesis of novel chiral stationary phases for high-performance liquid chromatography

Three novel chiral selectors 4a-c were synthesized from(S)-amino acids and(R)-1-phenyl-2-(4-methylphenyl)ethylamine.4a-cwere connected to 3-aminopropylsilanized silica gel to be used as the chiral stationary phase for HPLC.Five amino acid derivativesand two pyrethroid insecticides were fairly resolved on these three new chiral stationary phases under normal phase condition.     

Cationic cyclodextrins chemically-bonded chiral stationary phases for high-performance liquid chromatography

Two covalently bonded cationic β-CD chiral stationary phases (CSPs) prepared by graft polymerization of 6^A-(3-vinylimidazolium)-6-deoxyperphenylcarbamate-β-cyclodextrin chloride or 6^A-(N,N-allylmethylammonium)-6-deoxyperphenylcarbamoyl-β-cyclodextrin chloride onto silica gel were successfully applied in high-performance liquid chromatography (HPLC). Their enantioseparation capability was examined with 12 racemic pharmaceuticals and 6 carboxylic acids. The results indicated that imidazolium-containing β-CD CSP afforded more favorable enantioseparations than that containing ammonium moiety under normal-phase HPLC. The cationic moiety on β-CD CSPs could form strong hydrogen bonding with analytes in normal-phase liquid chromatography (NPLC) to enhance the analytes’ retention and enantioseparations. In reversed-phase liquid chromatography (RPLC), the analytes exhibited their maximum retention when the pH of mobile phase was close to their pK_a value. Inclusion complexation with CD cavity and columbic/ionic interactions with cationic substituent on the CD rim would afford accentuated retention and enantioseparations of the analytes.     

Polymer coatings as stationary phases in high-performance liquid chromatography

Modern biochromatography demands highly sophisticated packing materials in terms of biocompatibility, (substrate-) selectivity and recovery. Polymers can be designed in a wide variety and therefore deliver solutions to specific chromatographic problems. Thus, tailor-made polymer coatings are an alternative to the classical chemically bonded stationary phases.     

Novel stationary phases for high-performance liquid chromatography analysis of cyclodextrin derivatives

Novel stationary phases were prepared for separation of cyclodextrins and cyclodextrin derivatives by bonding substituted aromatic groups (phenyl and naphthyl) to the silica gel matrix. Both the electron-withdrawing (nitro) and the hydrogen-donor/acceptor (amide or carbamide) substituents of the phenyl group play essential role in the separation of cyclodextrins and cyclodextrin derivatives. On the basis of the comparison of experimental data obtained on different columns the N-(4-nitrophenyl)-carbamide group bonded silica gel stationary phase was selected as the most effective one for analysis of cyclodextrin derivatives. Improved separation potency was observed for hydroxypropylated, methylated and several other cyclodextrin derivatives compared with the previously and presently used stationary phases. Owing to the strong retention of cyclodextrins and its derivatives on the selected column, detection of their decomposition products was easily achieved. Determination of unsubstituted, natural cyclodextrin as an impurity in the cyclodextrin derivatives was implemented. Identification and characterization of cyclodextrin derivatives in industrial products could also be performed.     

New stationary phases for normal-phase high-performance liquid chromatography (NP-HPLC)

3-(2,3-Dihydroxy-propoxy)-propyl-silica (diolsilica) was modified to bonded stationary phases for normal-phase high-performance liquid chromatography (NPLC) carrying phosphorous ester, boronic ester, and nitric acid ester groups. The NPLC phases were investigated with respect to their properties towards separations of polyaromatic hydrocarbons, and chlorinated and nitrated environmental pollutants. Aminopropyl-silica and nitrooxy-propyl-silica (the latter synthesized in our working group) are presented for comparison. Depending on the functionality used for the modification the retention behavior can significantly be changed towards the three analyte classes. Especially 2,2,2-tribromoethyl-phosphoromorpholino-chloridate modified diol-silica, 2,2,2-tribromoethyl-dichlorophosphate modified diol-silica and nitrated diol-silica (nitrooxy-propoxypropyl-silica) show specific properties for the separation of polyaromatic hydrocarbons, chlorinated analytes and nitrated analytes. Preparation and packing of the adsorbents are described. Principal Component Analysis (PCA) is used as a chemometric tool for effective data reduction and visualization of the results in terms of retention behavior of the stationary phases. Received: 4 August 1997 / Revised: 3 December 1997 / Accepted: 7 December 1997     

Liquid chromatography of sugars on silica-based stationary phases

A review is given of sugar analysis by liquid chromatography using silica columns. Aspects covered are column materials and preparation, chemically and physically modified amine columns, octadecy- and unmodified silica columns; eluent composition and elution mechanisms for the different types of columns used; detection methods, RI and UV detectors, visible lights, fluorescence, moving-wire, polarimetric and mass detection; and sample preparation and origin of samples.     

Analysis of high-performance liquid chromatography bonded stationary phases using thermogravimetry

A thermogravimetric method was developed for determining the C-18 bonded phase content of reversed phase high performance liquid Chromatographic stationary phases. The method yielded data that were comparable to the sum of carbon and hydrogen content. Excellent agreement between the two methods was achieved by heating the stationary phase samples to 150°C in order to remove adsorbed species prior to elemental analysis.This research stemmed from the author's Master's degree research undertaken at California State Polytechnic University Pomona, and conducted at the Getty Conservation Institute (GCI).The author is greatly indebted to the following colleagues at the GCI for their advice during the course of this project: Neville Agnew, Charles Selwitz, Dusan Stulik and David Scott. The HPLC bonded stationary phase samples were prepared by John Streng, California State Polytechnic University, Pomona.     

Chloromethylphenylcarbamate derivatives of cellulose as chiral stationary phases for high-performance liquid chromatography

A new class of eight chloromethylphenylcarbamate derivatives of cellulose was prepared by introducing both an electron-donating methyl group and an electron-withdrawing chloro group on to the phenyl moieties and their chiral recognition abilities were evaluated as chiral stationary phases (CSPs) for high-performance liquid chromatography. The superiority of these derivatives over dichloro- and dimethylphenylcarbamates of cellulose as CSPs was demonstrated for some racemic compounds. The elution order and enantioselectivity were greatly dependent on the positions of the substituents. Meta- and para-disubstituted derivatives showed higher chiral recognition than ortho- and meta- or para- disubstituted derivatives. The correlation between the chemical shifts of the N---H protons of the carbamate moieties and the enantiomer-resolving abilities of the derivatives is discussed. Some of the derivatives were effective CSPs in both normal- and reversed- phase conditions and could efficiently separate some chiral drug enantiomers.     

Atypical silica-based column packings for high-performance liquid chromatography

Column packings widely used for high-performance liquid chromatography (HPLC) mostly are based on porous silica microspheres with certain pore sizes and pore size distributions. Such materials have the most desirable compromise of properties that provide for effective and reproducible separations over a wide range of operating conditions. To provide desired separation characteristics, several manufacturers specially synthesize the silica particles for these packings. While such column packing materials have general utility for a wide range of needs, special silica-based particles have been synthesized with different physical conformations for special separation goals. This presentation describes some atypical types of silica-based particles with unique separation properties that enlarge the capabilities of HPLC methods.     

Immobilized polysiloxanes as stationary phases for high-performance liquid chromatography and solid phase extraction

Polysiloxanes immobilized onto the surfaces of porous silica particles have proven to be good stationary phases for the separation of multiresidues of pesticides and their metabolic/degradation products by reversed-phase high-performance liquid chromatography (RP-HPLC). Similar materials have proven effective for pre-concentration and clean-up procedures using solid phase extraction. The present paper describes the preparation and some applications of several of these packing materials.     

Ionic liquid stationary phases for gas chromatography

This article provides a summary of the development of ionic liquids as stationary phases for gas chromatography beginning with early work on packed columns that established details of the retention mechanism and established working methods to characterize selectivity differences compared with molecular stationary phases through the modern development of multi-centered cation and cross-linked ionic liquids for high-temperature applications in capillary gas chromatography. Since there are many reviews on ionic liquids dealing with all aspects of their chemical and physical properties, the emphasis in this article is placed on the role of gas chromatography played in the design of ionic liquids of low melting point, high thermal stability, high viscosity, and variable selectivity for separations. Ionic liquids provide unprecedented opportunities for extending the selectivity range and temperature-operating range of columns for gas chromatography, an area of separation science that has otherwise been almost stagnant for over a decade.     

Chiral high-performance liquid chromatography of aromatic cyclic dipeptides using cyclodextrin stationary phases

A series of enantiomers of cyclic and linear dipeptides containing aromatic amino acids was prepared and chromatographed on beta- and gamma-cyclodextrin (CD) columns. The retention times, separation factor alpha and resolution values were calculated. The relevance of the distance of the chiral center from the phenyl ring for chiral resolution was studied. A model was developed using X-ray crystallographic data for an inclusion complex of beta-CD and the enantiomers of cyclic (Phe-Gly).     

Fluorocarbon stationary phases for liquid chromatography applications

This article presents an overview on fluorocarbon stationary phases for liquid chromatography (LC) applications. Fluorocarbons developed as alternative reverse phases have revealed previously unknown separation mechanisms and special utilities. Solvophobicity and fluorophilicity of the fluorinated phases provide enhanced selectivity for organofluorine compounds. The dual normal- and reverse-phase characteristics make fluorinated phases suitable for analysis of polar pharmaceutical and biological samples such as proteins, peptides, nucleotides, steroids, and alkaloids. Fluorinated phases for other applications including supercritical fluid chromatography (SFC), micellar electrokinetic liquid chromatography (MEKC), ion chromatography (IC), open tubular electrochromatography (OTEC), and liquid chromatography-mass spectrometry (LC-MS) are also highlighted.     

Synthesis and characterization of new organometallic complexes bonded stationary phases for high-performance liquid chromatography

Summary The preparation, characterization and potential liquid chromatographic applications of various organometallic iron complexes silica stationary phases are presented. These new supports are synthesized by covalently linking ferrocene, as well as some of its cationic derivatives, to appropriately derivatized silica support matrices. These columns exhibit moderate to high selectivity towards the separation of polycyclic aromatic hydrocarbons (PAHs). A charge transfer retention mechanism has been proposed. A comparison with a reference stationary phase, 3,5-dinitrobenzamide (DNB), to quantify the acceptor power of the new stationary bonded phases, is also reported. Finally, the effect of varying the derivatives of the bonded metallocene on PAHs retention is discussed.     

Covalently bonded polysaccharide derivatives as chiral stationary phases in high-performance liquid chromatography

Polysaccharide derivatives have been extensively used as chromatographic chiral selectors in chiral stationary phases (CSPs) for the separation of enantiomers by HPLC. When coated onto a silica matrix, they represent nowadays one of the most popular type of CSPs. However, they are only compatible with a limited choice of solvents. The main drawback of these CSPs is related to the solubility of the chiral selector in a number of solvents, which limits their applicability. The different attempts which have been described up to now to overcome this problem by covalently fixing the chiral selector to a matrix are reviewed in this paper.