Monolithic stationary phases for liquid chromatography and capillary electrochromatography

A monolithic stationary phase is the continuous unitary porous structure prepared by in situ polymerization or consolidation inside the column tubing and, if necessary, the surface is functionalized to convert it into a sorbent with the desired chromatographic binding properties [J. Chromatogr. A 855 (1999) 273]. Monolithic stationary phases have attracted considerable attention in liquid chromatography and capillary electrochromatography in recent years due to their simple preparation procedure, unique properties and excellent performance, especially for separation of biopolymers. This review summarizes the preparation, characterization and applications of the monolithic stationary phases. In addition, the disadvantages and limitations of the monolithic stationary phases are also briefly discussed.     

Monolithic capillary columns for liquid chromatography on the basis of ethylene glycol dimethacrylate

The effects of the synthesis conditions and polymerization mixture composition on the characteristics of monolithic capillary columns prepared from ethylene glycol dimethacrylate were studied. It was demonstrated that the content of the monomer in the initial mixture only slightly influences the efficiency of the columns obtained but, at the same time, causes a significant change in their separation impedance. By contrast, the temperature of synthesis strongly affects both these parameters. The efficiency of monolithic capillary columns prepared under optimum conditions was found to correspond to that of columns packed with a 5-μm-grained sorbent; however, the separation impedance of the former is substantially lower. A typical chromatogram of separation of a test mixture is presented. Original Russian Text ? A.N. Kanat’eva, A.A. Korolev, E.N. Viktorova, A.A. Kurganov, 2007, published in Zhurnal Fizicheskoi Khimii, 2007, Vol. 81, No. 3, pp. 568–572.     

Monolithic silica columns with various skeleton sizes and through-pore sizes for capillary liquid chromatography

Reduction of through-pore size and skeleton size of a monolithic silica column was attempted to provide high separation efficiency in a short time. Monolithic silica columns were prepared to have various sizes of skeletons (approximately 1-2 microm) and through-pores (approximately 2-8 microm) in a fused-silica capillary (50-200 microm I.D.). The columns were evaluated in HPLC after derivatization to C18 phase. It was possible to prepare monolithic silica structures in capillaries of up to 200 microm I.D. from a mixture of tetramethoxysilane and methyltrimethoxysilane. As expected, a monolithic silica column with smaller domain size showed higher column efficiency and higher pressure drop. High external porosity (> 80%) and large through-pores resulted in high permeability (K = 8 x 10(-14) -1.3 x 10(-12) m2) that was 2-30 times higher than that of a column packed with 5-mirom silica particles. The monolithic silica columns prepared in capillaries produced a plate height of about 8-12 microm with an 80% aqueous acetonitrile mobile phase at a linear velocity of 1 mm/s. Separation impedance, E, was found to be as low as 100 under optimum conditions, a value about an order of magnitude lower than reported for conventional columns packed with 5-microm particles. Although a column with smaller domain size generally resulted in higher separation impedance and the lower total performance, the monolithic silica columns showed performance beyond the limit of conventional particle-packed columns under pressure-driven conditions.     

Porous ceramic bed supports for fused silica packed capillary columns used in liquid chromatography

Porous ceramic bed supports for fused silica packed capillary columns utilized in liquid chromatography were prepared by polymerizing solutions containing potassium silicate in-situ within a column to create a mechanically stable, rugged, and easily constructed termination. The effect of the bed support length on efficiency, and comparisons to glass wool bed supports, were considered in terms of column efficiencies and hydrodynamic variables. Results obtained indicate better performance for the ceramic bed support.     

Monolithic columns in high-performance liquid chromatography

Monolithic media have been used for various niche applications in gas or liquid chromatography for a long time. Only recently did they acquire a major importance in high-performance column liquid chromatography (HPLC). The advent of monolithic silica standard- and narrow-bore columns and of several families of polymer-based monolithic columns has considerably changed the HPLC field, particularly in the area of narrow-bore columns. The origin of the concept, the differences between their characteristics and those of traditional packed columns, their advantages and drawbacks, the methods of preparation of monoliths of different forms, and the current status of the field are reviewed. The actual and potential performance of monolithic columns are compared with those of packed columns. Monolithic columns have considerable advantages, which makes them most useful in many applications of liquid chromatography. They are extremely permeable and offer a high efficiency that decreases slowly with increasing flow velocity.     

Packed capillary columns for liquid chromatography

Summary Fused silica capillaries, ≈ 130 × 0.32mm have been packed with small reversed phase spherical silica particles, 3 or 2μm, in order to achieve LC-systems giving high plate numbers at relatively low pressure drop in short analysis times. At optimal conditions, columns packed with 3μm particles showed reduced plate heights, h, around 2.5, and the column flow resistance, ϕ, was 335–625. With 2μm particles, a reduced plate height of only 3.7 was achieved, which reflects the difficulty in the packing of such small particles.     

Monolithic silica rod columns for high-efficiency reversed-phase liquid chromatography

Chromatographic properties of a new type of monolithic silica rod columns were examined. Silica rod columns employed for the study were prepared from tetramethoxysilane, modified with octadecylsilyl moieties, and encased in a stainless-steel protective column with two polymer layers between the silica and the stainless-steel tubing. A 25 cm column provided up to 45,000 theoretical plates for aromatic hydrocarbons, or a minimum plate height of about 5.5 μm, at optimum linear velocity of ca. 2.3 mm/s and back pressure of 7.5 MPa in an acetonitrile-water (80/20, v/v) mobile phase at 40°C. The permeability of the column was similar to that of a column packed with 5 μm particles, with K(F) about 2.4×10(-14) m(2) (based on the superficial linear velocity of the mobile phase), while the plate height value equivalent to that of a column packed with 2.5 μm particles. Generation of 80,000-120,000 theoretical plates was feasible with back pressure below 30 MPa by employing two or three 25 cm columns connected in series. The use of the long columns enabled facile generation of large numbers of theoretical plates in comparison with conventional monolithic silica columns or particulate columns. Kinetic plot analysis indicates that the monolithic columns operated at 30 MPa can provide faster separations than a column packed with totally porous 3-μm particles operated at 40 MPa in a range where the number of theoretical plates (N) is greater than 50,000.     

Polymethacrylate monolithic columns for capillary liquid chromatography

In recent years, continuous separation media have attracted considerable attention because of the advantages they offer over packed columns. This research resulted in two useful monolithic material types, the first based on modified silica gel and the second on organic polymers. This work attempts to review advances in the development, characterization, and applications of monolithic columns based on synthetic polymers in capillary chromatography, with the main focus on monolithic beds prepared from methacrylate-ester based monomers. The polymerization conditions used in the production of polymethacrylate monolithic capillary columns are surveyed, with attention being paid to the concentrations of monomers, porogen solvents, and polymerization initiators as the system variables used to control the porous and hydrodynamic properties of the monolithic media. The simplicity of their preparation as well as the possibilities of controlling of their porous properties and surface chemistries are the main benefits of the polymer monolithic capillary columns in comparison to capillary columns packed with particulate materials. The application areas considered in this review concern mainly separations in reversed-phase chromatography, ion-exchange chromatography, hydrophobic and hydrophilic interaction modes; enzyme immobilization and sample preparation in the capillary chromatography format are also addressed.     

On-line multidimensional chromatography using packed capillary liquid chromatography and capillary gas chromatography

The introduction of selected fractions from a liquid chromatograph into a gas chromatograph has been described; however, analyses were performed by off-line experiments requiring collection and reinjection of the separate fractions or by on-line procedures where disadvantageously, only a fraction of the separated peak or a well resolved component in a mixture could be introduced into a gas chromatograph. This disadvantage is overcome by the apparatus and method described in this paper, which utilizes a multidimensional chromatography system employing a high efficiency, packed capillary LC column coupled on-line to a capillary gas chromatograph. The liquid chromatograph (so designed) can act as a highly efficient clean-up or chemical class fractionation step prior to introduction into the gas chromatograph, significantly reducing sample preparation times in many applications. Thus minor components in a complex matrix can be determined without prior sample clean-up, an example of which is the determination of polychlorinated biphenyls in a complex hydrocarbon matrix.     

Non-particulate (continuous bed or monolithic) acrylate-based capillary columns for reversed-phase liquid chromatography and electrochromatography

Three approaches are described to synthesize acrylic non-particulate beds (also called continuous beds or monoliths) in aqueous polymerization media for reversed-phase capillary liquid chromatography/electrochromatography. In the first, hexyl acrylate comonomer was dissolved together with water soluble polar comonomers using a non-ionic detergent. In the second, a new alkyl ammonium salt comonomer, (3-allylamino-2-hydroxypropyl)dodecyldimethylammonium chloride was used, which is water soluble and has detergent properties itself. The alkyl group of this comonomer provides hydrophobicity while the ionic groups generate electroosmosis in the non-particulate bed. In the third approach, the alkyl comonomer was used as a detergent to dissolve another hydrophobic comonomer in an aqueous polymerization medium. All three approaches were evaluated with respect to hydrophobicity, efficiency and electroosmotic properties of the beds. Hydrophobicity expressed as methylene group selectivity for the three types of the beds in 50% methanol mobile phase was 1.86, 1.16 and 1.78, electroosmotic mobility -5.14 x 10(-5), 6.89 x 10(-5) and 6.37 x 10(-5) cm2 V(-1) s(-1) and efficiency for the retained compound (methylparabene) 67,000, 93,000 and 110,000 plates m(-1) correspondingly. The columns were tested using pressure driven capillary chromatography and capillary electrochromatography. The influence of polymerization temperature on hydrodynamic permeability, separation impedance and inverse size exclusion porosimetry characteristics were used to evaluate the separation columns. The increase of the polymerization temperature resulted higher permeability of the bed, separation impedance and lower polymeric skeleton porosity. Further characterisation was provided by examining the separation efficiency observed for a series of benzoic acid esters and alkyl parabens.     

Monolithic columns based on a poly(styrene-divinylbenzene-methacrylic acid) copolymer for capillary liquid chromatography of small organic molecules

A very simple and readily performed method is described for the preparation of poly(styrene-divinylbenzene-methacrylic acid) monolithic columns for capillary liquid chromatography. The effect of the methacrylic acid content on the morphological and chromatographic properties has been investigated. Methacrylic acid is shown to be essential for isocratic separations of small organic analytes by capillary liquid chromatography. Column efficiencies of about 28,000 theoretical plates/m have been obtained for all the test compounds. The batch-to-batch and run-to-run repeatability of the retention times is better than 1.5%.     

Divinylbenzene-based monolithic capillary columns in capillary liquid chromatography

The effect of the conditions of synthesis of divinylbenzene-based monolithic capillary columns on their chromatographic characteristics was studied. It was demonstrated that the porosity and permeability of the column change significantly even at small deviations from the optimum conditions of polymerization of the monolith in the column. By contrast, the minimum value of HETP proved to be only slightly sensitive to the conditions of synthesis, ranging within ～10–20 μm. The conditions of polymerization of the monolith were found to produce more pronounced effect on the slope of the right branch of the van Deemter curve (parameter C), with the flattest curve being observed for columns prepared under optimum conditions. The minimum value of HETP for polymer monolithic capillary columns was found to be similar to that for silica gel monolithic capillary columns, but the latter are characterized by C values approximately an order of magnitude lower.     

Anion exchange silica monolith for capillary liquid chromatography

An anion exchange monolithic silica capillary column was prepared by surface modification of a hybrid monolithic silica capillary column prepared from a mixture of tetramethoxysilane (TMOS) and methyltrimethoxysilane (MTMS). The surface modification was carried out by on-column copolymerization of N-[3-(dimethylamino)propyl]acrylamide methyl chloride-quaternary salt (DMAPAA-Q) with 3-methacryloxypropyl moieties bonded as an anchor to the silica surface to form a strong anion exchange stationary phase. The columns were examined for their performance in liquid chromatography (LC) and capillary electrochromatography (CEC) separations of common anions. The ions were separated using 50 mM phosphate buffer at pH 6.6. Evaluation by LC produced an average of 30,000 theoretical plates (33 cm column length) for the inorganic anions and nucleotides. Evaluation by CEC, using the same buffer, produced enhanced chromatographic performance of up to ca. 90,000 theoretical plates and a theoretical plate height of ca. 4 μm. Although reduced efficiency was observed for inorganic anions that were retained a long time, the results of this study highlight the potential utility of the DMAPAA-Q stationary phase for anion separations. Figure Micro-LC performance evaluation of a strong anion exchange silica monolith column, 100H-MOP-DMAPAA-Q, 33 cm in length, with a mobile phase of 50 mM phosphate buffer, pH 2.8; linear velocity: u = 1.8 mm/s; UV-Vis detection at 254 nm. Sample solution (5 mg/mL of each component, 4 mL) was injected in split flow injection mode at a split ratio of ca. 1:1900 with a pump flow rate of 1.5 mL/min     

Tailoring the macroporous structure of monolithic silica-based capillary columns with potential for liquid chromatography

The present work aims at the optimisation of the synthesis of methyl-silsesquioxane monolithic capillary columns using a sol-gel based protocol. The influence of reaction conditions such as temperature, reaction mixture composition and catalyst concentration has been examined. The morphology of the products was studied by scanning electron microscopy and nitrogen adsorption. Monolithic capillary columns were obtained with a skeleton-like structure with open pores. Pore diameters vary from 0.8 to 15 microm, diameters of the xerogel network vary from 0.4 to 12 microm, respectively. Specific surface areas up to 334 m2/g have been observed, however, many materials did not possess areas above few m2/g which represents the limit of detection of the nitrogen porosimetry measurements. Excellent adhesion to the capillary wall was observed in all cases, and drying was possible at ambient conditions without the formation of cracks.     

An electroosmotic pump for packed capillary liquid chromatography

An electroosmotic pump (EOP) capable of generating pressure above 3 MPa and μl/min flow rate with reverse phase mobile phases of HPLC was constructed and evaluated. The pump consisted of three parallel connected fused silica capillary columns (25 cm×320 μm I.D.) packed with 2 μm silica materials, hollow electrodes, a high voltage DC power supply, and a liquid pressure transducer. The EOP was applied in a capillary liquid chromatographic system for mobile phase delivery instead of a mechanical pump. Standard samples containing thiourea, naphthalene, anthracene, phenanthrene and acetonitrile were separated on a 15 cm×320 μm I.D. 5 μm Chromasil C₁₈ packed capillary column with acetonitrile/water as mobile phase.     

Monolithic stationary phases for fast ion chromatography and capillary electrochromatography of inorganic ions

The focus of this review is on current developments in monolithic stationary phases for the fast analysis of inorganic ions and other small molecules in ion chromatography (IC) and capillary electrochromatography (CEC), concentrating in particular on the properties of organic (polymer) monolithic materials in comparison to inorganic (silica-based) monoliths. The applicability of these materials for fast IC is discussed in the context of recent publications, including the range of synthesis and modification procedures described. While commercial monolithic silica columns already show promising results on current IC instrumentation, polymer-based monolithic stationary phases are currently predominantly used in the capillary format on modified micro-IC systems. However, they are beginning to find application in IC particularly under high pH conditions, with the potential to replace their particle-packed counterparts.