Porous Monoliths: Stationary Phases of Choice for High Performance Liquid Chromatography in Various Formats

 Modern porous monoliths have been conceived as a new class of stationary phases for high performance liquid chromatography (HPLC) in classical columns in the early 1990s and later extended to the capillary format. These monolithic materials are prepared using simple processes carried out in an external mold (inorganic monoliths) or within the confines of the column (organic monoliths and all capillary columns). These methods afford macroporous materials with large through-pores that enable applications in a rapid flow-through mode. Since all the mobile phase must flow through the monolith, the convection considerably accelerates mass transport within the monolithic separation medium and improves the separations. As a result, the monolithic columns perform well even at very high flow rates. The applications of monolithic capillary columns are demonstrated on numerous separations in the HPLC mode.     

Liquid phase separation methods: HPLC,FFF, electrophoresis

Summary Recent research in separations has been directed towards extending the molecular weight range for these high-resolution methods, while at the same time improving reproducibility and convenience of operation. This overview summarizes some of these developments and provides insights into the inherent resolution, range, speed, and limitations of these methods. Important advances in HPLC include the development of better siliceous supports, as well as fresh approaches for producing both covalently-bonded and crosslinked, mechanically-immobilized organic stationary phases that provide a new level of reproducibility and stability for separations. Very wide-pore packings now permit separations of species with molecular weights up to about 10⁷ daltons, and columns of particles in the 1–2 μm range allow rapid separations of macromolecules. New results in sedimentation and thermal field flow fractionation (FFF) demonstrate the power of these methods to separate species with high resolution in molecular weight ranges that cannot be accessed by either HPLC or electrophoresis. Of special interest is the use of sedimentation FFF for isolating soluble, high-molecular-weight molecules and particulates such as sub-cellular components. Recent studies have confirmed that thermal FFF is a powerful method for determining the molecular-weight distribution of fragile, high-molecular-weight synthetic polymers with good accuracy and reproducibility. Capillary electrophoresis techniques give promise of very fast, high-resolution separations of ionic macromolecules; on-line detection affords high sensitivity, and quantitation is readily achievable. Special agarose gel electrophoretic techniques now permit excellent DNA separations for species up to 5 × 10⁶ base pairs, and 2D electrophoretic methods provide resolution of more than 10⁴ components in coupled sequential separations of protein mixtures.     

Egg white protein-bonded columns and their applications.

 Egg white protein bonded columns were developed for HPLC. These columns can be used under aqueous mobile phase and separate various kinds of drug enantiomers. Hyphenated techniques using protein bonded columns with LC/MS and/or column switching have been recognized as integral methods in pharmaceutical research to analyze drug enantiomers. Therefore, these methods are very useful for research fields of pharmacokinetics and pharmacology.     

A practical approach to the preparative purification of peptides using analytical instrumentation with analytical and semipreparative columns

Summary We have examined the effect of particle size of silica-based reversed-phase packings and column packing techniques on the reversed-phase analytical separation of a peptide mixture. A C₁₈ packing of 15–20 μm average particle size produced satisfactory peptide resolution, allowing a relatively inexpensive scale up to the preparative purification of peptides. A shallow gradient (0.1% acetonitrile/min) elution procedure was developed for the preparative purification of closely related decapeptides (differing by one methyl group) on analytical (250×4.1 mm I.D.) and semipreparative (250×10 mm I.D.) columns. Up to 30 mg and 225 mg of the two-peptide mixture was efficiently resolved, with high yields of homogeneous peptides, on analytical and semipreparative columns, respectively, containing the 15–20 μm packing. We have also demonstrated the potential of our purification procedure for resolving more complex multicomponent mixtures by efficiently separating a total of 22 mg of three closely-related peptides on analytical columns containing 7 μm or 15–20 μm particle size reversed-phase packings. The use of the inexpensive 15–20 μm packing, coupled with the ability to pack efficient columns with analytical HPLC instrumentation, offers great cost saving potential.     

THE MOLECULAR WEIGHT DEPENDENCE OF THE SPREADING FACTOR OF SEC COLUMNS AND THE SURFACE STRUCTURE OF THE PORE OF PACKINGS

The molecular weight separation and column dispersion for a number of SEC columns packedwith various packing materials were calibrated simultaneously using narrow MWD polystyrene stan-dards as probe. The molecular weight dependence of the spreading factor is closely related to thesurface structure in the pore of packings.     

Advances in Capillary Chromatography

  Abstract：Capillary columns are used in both capillary liquid chromatography and capillary electrochromatography. The design for capillary liquid chromatography is discussed in comparison with capillary gas chromatography. The difference of diffusion coefficient in gas and liquid phase is a key role. The study for obtaining a high performance capillary liquid chromatography is discussed. Capillary electrochromatography is recently interesting for its instinct ability to realize a high performance chromatography. Capillary electrochromatography with and without pressurized flow is reviewed briefly. Instrumentation for capillary electrochromatography with pressurized flow is discussed. The port of splitting, and gradient elution of both solution and potential are described. The new findings of both the variation of column resistance and capacity factor according to the value of applied electric voltage are also discussed.     

“Click chemistry” preparation of WCX packings for protein separation

Click chemistry was applied to immobilize three kinds of alkyne-carboxylic acids onto azide-modified silica gel to prepare three novel stationary phases for weak cation exchange chromatography(WCX).The developed protocol combines the benefits of operational simplicity,exceptionally mild conditions and high surface loadings.Six kinds of standard proteins were separated completely on the novel packings.Compared with commercial WCX columns,the three kinds of novel WCX packings prepared by click chemistry approach have better resolution and selectivity.Lysozyme was purified successfully from egg white with the novel WCX column by one step.The purity was more than 97%and a high specific activity was achieved to be 81,435 U/mg.The results illustrate the potential of click chemistry for preparation of stationary phase for IEC.     

一种新的分离三硝基甲苯及其代谢产物的高效液相色谱方法

An HPLC method is described for separation of TNT and its main reduction metabolites. Two columns(LC-C(18) and LC-CN)are connected in series and operated isocratically at 1.0mL/min with water-methanol-tetrahydrofuran(60+ 35+ 5 ). The baseline separation of 4-amino-2,6-dinitrotoluene(4A) and 2-amino-4,6-dinitrotoluene(2A) ,which are main metabolites of TNT,is obtained(Rs=2.1).     

Fast headspace analysis with short microcapillary columns

Summary A new method of analysis using headspace gas chromatography with microcapillary columns is proposed. Small diameter (50 μm I.D.) fused-silica capillary columns with non-extractable SE-54 and PS-255 polysiloxane stationary phases were used for the analysis of low boiling organic compounds. The small diameter columns possess the usual very high efficiency so that the method can be employed for the headspace analysis of complex mixtures. The use of short microcolumns reduces the analysis times in comparison to conventional capillary columns. Good performances were obtained in the analysis of volatile compounds in some lemon essential oil, perfumes, and water samples.     

Click chemistry: a new facile and efficient strategy for preparation of functionalized HPLC packings

Click chemistry has been successfully extended into the field of preparation of functionalized HPLC packings, proving a novel facile and efficient strategy for covalently bonding stationary phases onto HPLC grade silica beads; the potential has been demonstrated by the preparation of "Click I-IV" columns and preliminary results in the separation of sugars.     

Development of a so-called hour method for analysis of plutonium in soil samples

A short overview of HPLC column packings is presented. The properties of chromatographic carriers and the possibilities to combine the solid matrices with organic polymeric stationary phases are elucidated in detail. The latest achievements and anticipated future developments in the area are outlined.     

Solvent delivery in the new application areas of high-performance liquid chromatography

Summary The development of new column packings for biological macromolecules, as well as the techniques for handling micropacked columns, sets new demands on solvent delivery systems for HPLC. The main implications of these developments with respect to pump design are discussed and the evaluation of a solvent delivery system (pump and gradient programmer) optimized for these purposes is presented.Presented at the 14th International Symposium on Chromatography London, September, 1982     

高效液相色谱用硅质填料的进展

 对高效液相色谱用硅质填料的进展情况做了全面的评述。介绍了硅质填料的制备及化学修饰 ,评价了硅胶表面的物理表征 ,概括了硅质填料在高效液相色谱中的应用 ,并对我国的硅质填料前景进行了展望。     

HPLC of basic drugs using non‐aqueous ionic eluents: evaluation of a 3 μm strong cation‐exchange material

HPLC columns packed with 3 μm particle size HPLC Technology Techsphere SCX (propylsulfonic acid‐modified) silica offer considerable advantages over 5 μm SCX packings in the analysis of basic drugs using 100% methanol eluents containing an ionic modifier such as ammonium perchlorate. The basic drugs studied included clozapine and norclozapine, olanzapine, quinine and quinidine, and amitriptyline, nortriptyline, imipramine and desipramine. The 3 μm column was not only more efficient for a given column length compared with 5 μm materials, but also elution times were less, a phenomenon observed in reversed‐phase systems. The high efficiencies and excellent peak shapes obtained with the 3 μm SCX‐modified packing together with the relatively low back‐pressures attained show that such materials deserve serious consideration by laboratories involved in the analysis of basic drugs. Manufacturers should offer such packings as a matter of routine. Alternative ionic modifiers such as ammonium acetate are available for use with mass spectrometric detection if required. Copyright © 2009 John Wiley & Sons, Ltd.     

Polymeric columns for liquid chromatography

Polymeric columns are becoming used more frequently in modern liquid chromatography applications as improvements in polymeric packing materials are realized and as more applications are developed for these materials. Modern polymers have overcome earlier problems associated with their use; higher rigidity now allows them to be used at normal eluent flow rates resulting in faster analyses, and improved synthesis techniques have resulted in efficiencies comparable to the best silica materials, sometimes exceeding 100,000 plates/m. In addition, polymers offer distinct advantages over silica packing for particular applications. Because silica packings are readily degraded by aqueous eluents, they are not always suitable for separations involving ionic species. Polymeric columns are particularly useful in determinations of amino acids, peptides, proteins, organic acids, carbohydrates, and inorganic cations and anions. Polymers are also characterized by exceptional lot-to-lot reproducibility. Reproducibility is often problematic with silica packings, particularly the bonded phases.     

Influence of the Structure of Chemically Bonded C 18 Phases on HPLC Separation of Naproxen Glucuronide Diastereoisomers

Abstract

On the basis of Nucleosil-100 a series of materials with varying structure and different coverage density of chemically bonded C₁₈ phases (CBP) were prepared. The physico-chemical characteristics of these packings e.g. porosity and carbon content were studied by the BET method and CHN analysis. The structure of the C₁₈ CBP was determined by solid state CP/MAS NMR. The prepared packings and columns have been applied for HPLC separation of naproxen and its two diastereoisomeric conjugates with glucuronic acid. Material with monomeric C₁₈ CBP structure and with high coverage density has given good and reproducible separation results.     

Fundamental and practical aspects of ultrahigh pressure liquid chromatography for fast separations

The ongoing development of HPLC has been focused on increasing the speed and efficiency of separations over the past decade. The advances in separation speed have been primarily related to the development of column technology and instrumentation. Relatively short columns packed with sub-2 microm particles provide high-speed separations while maintaining or increasing resolution. Ultrahigh pressure pump systems have been developed to overcome the high-pressure drop generated by such sub-2 microm packings. In this review, fundamental and practical aspects of ultrahigh pressure or ultrahigh performance liquid chromatography (U-HPLC) are discussed. Applications of fast U-HPLC separations are also presented.     

Characterization of Column Packings in Normal-Phase Liquid Chromatography. II. Retention Behavior of Fat-Soluble Vitamins in Amino- and Cyano-Propyl Bonded Silica Gel and Binary Solvent Systems

Abstract

As continuation of our study on the characterization of column packings in normal-phase HPLC analysis, the retention indices of ten fat-soluble vitamins on aminopropyl and cyanopropyl bonded silica columns were systematically estimated using binary solvents containing ethyl acetate (EtOAc), tetrahydrofuran (THF) or 2-propanol (PrOH) in n-hexane. A linear relationship between the logarithm of the capacity ratio and that of the solvent composition was confirmed. The retentivity and selectivity for these chemically bonded packing columns were determined as follows: the amino-type column has stronger and cyano-type column weaker retentivity than the bare silica column for n-hexane-EtOAc or THF binary systems. Specific adsorption of tocopherol derivatives containing phenolic hydroxyl groups on the amino column was observed. To obtain high efficiency in the separation of fat soluble vitamins, peak sharpness and asymmetry factors were measured using three columns and three binary solvents. The bare silica and PrOH binary solvent generally gave superior peak shape for all vitamin samples.     

Preparation of Immobilized Metal Affinity Chromatographic Packings Based on Monodisperse Hydrophilic Non‐porous Beads and Their Application

Three hydrophilic immobilized metal affinity chromatographic packings for HPLC have been synthesized by chemical modification of 3.0 µm monodisperse non‐porous poly(glycidyl methacrylate‐co‐ethylenedimethacrylate) (P_GMA/EDMA) beads. The retention behavior of proteins on the metal ion chelated columns loaded with copper(II), nickel(II) and zin(II) ion was studied. The effect of pH on the protein retention was investigated on both the naked and metal ion chelated columns in the range from 4.0 to 9.0. Four proteins were quickly separated in 3.0 min with linear gradient elution at a flow rate of 3.0 mL/min by using the synthesized Ni²⁺‐IDA (iminodiacetic acid) packings. The separation time was shorter than other immobilized metal affinity chromatography reported in the literature. Purification of lysozyme from egg white and trypsin on the commercially available trypsin was performed on the naked‐IDA and Cu²⁺‐IDA columns, respectively. The purities of the purified trypsin and lysozyme were more than 92% and 95%, respectively.