Octyl-type monolithic columns of 530 microm i.d. for capillary liquid chromatography

A novel monolithic capillary column (530 microm i.d.) was prepared for capillary liquid chromatography (CLC) by in situ copolymerization of octyl methacrylate (MAOE) and ethylene dimethacrylate (EDMA) in the presence of a porogen solvent containing 1-propanol, 1,4-butanediol, and water with azobisisobutyronitrile as the initiator. The influences of the contents of the porogen solvent, EDMA and the various concentration ratios of 1-propanol to 1,4-butanediol in the polymerization mixture on the morphology, porosity, globule size, stability and column efficiency were investigated. The morphology and pore size distribution of monolithic capillary columns were characterized by SEM and mercury intrusion porosimetry, respectively. Chromatographic evaluations of the columns were performed under CLC mode. The results showed that good permeability and stability can be obtained under optimal experimental conditions. The separation results of some acid, neutral and basic analytes demonstrated the hydrophobicity and low affinity to basic analytes of the new column. Three metal ions, i.e. Mg(II), Zn(II) and Cd(II) were also separated under ion-pair mode on the new monolithic capillary column and the results were acceptable.     

The effect of hydrothermal treatment on column performance for monolithic silica capillary columns

Monolithic silica capillary columns with i.d. 100 μm and monolithic silica rods were prepared with tetramethoxysilane (TMOS) or a mixture of TMOS and metyltrimethoxysilane (MTMS) using different hydrothermal treatments at T=80 °C or 120 °C. Nitrogen physisorption was applied for the pore characterization of the rods and inverse size exclusion chromatography (ISEC) for that of the capillary columns. Using nitrogen physisorption, it was shown change of pore size and surface area corresponds to that of hydrothermal treatment and silica precursor. The results from ISEC agreed well with those from nitrogen physisorption regarding the pore size distribution (PSD). In addition, the retention factors for hexylbenzene with the ODS-modified capillary columns in methanol/water=80/20 at T=30 °C could also support the results from nitrogen physisorption. Furthermore, column efficiency for the columns was evaluated with alkylbenzenes and three kinds of peptides, leucine-enkephalin, angiotensin II, and insulin. Column efficiency for alkylbenzenes was similar independently of the hydrothermal treatment at T=120 °C. Even for TMOS columns, there was no significant difference in column efficiency for the peptides despite the difference in hydrothermal treatment. In contrast, for hybrid columns, it was possible to confirm the effect on hydrothermal treatment at T=120 °C resulting in a different column efficiency, especially for insulin. This difference supports the results from both nitrogen physisorption and ISEC, showing the presence of more small pores of ca. 3-6 nm for a hybrid silica without hydrothermal treatment at T=120 °C. Consequently, the results suggest that hydrothermal treatment for a hybrid column with higher temperature or longer time is necessary, compared to that for a TMOS column, to provide higher column efficiency with increase in molecular size of solute.     

Properties of monolithic silica columns for HPLC.

Monolithic silica columns and their use in high peak-capacity HPLC separations are reviewed. Monolithic silica columns can potentially provide higher overall performance than particle-packed columns based on the variable external porosity and variable through-pore size/skeleton size ratios. The high permeability of monolithic silica columns resulting from the high porosity is shown to be advantageous to generate large numbers of theoretical plates with long capillary columns. High permeability together with the high stability of the network structures of silica allows their use in high-speed separations required for a second-dimension column in two dimensional HPLC. Disadvantages of monolithic silica columns are also described.     

Comparison of monolithic silica and polymethacrylate capillary columns for LC

Organic polymer monolithic capillary columns were prepared in fused-silica capillaries by radical co-polymerization of ethylene dimethacrylate and butyl methacrylate monomers with azobisisobutyronitrile as initiator of the polymerization reaction in the presence of various amounts of porogenic solvent mixtures and different concentration ratios of monomers and 1-propanol, 1,4-butanediol, and water. The chromatographic properties of the organic polymer monolithic columns were compared with those of commercial silica-based particulate and monolithic capillary and analytical HPLC columns. The tests included the determination of H-u curves, column permeabilities, pore distribution by inversed-SEC measurements, methylene and polar selectivities, and polar interactions with naphthalenesulphonic acid test samples. Organic polymer monolithic capillary columns show similar retention behaviour to chemically bonded alkyl silica columns for compounds with different polarities characterized by interaction indices, Ix, but have lower methylene selectivities and do not show polar interactions with sulphonic acids. The commercial capillary and analytical silica gel-based monolithic columns showed similar selectivities and provided symmetrical peaks, indicating no significant surface heterogeneities. To allow accurate characterization of the properties of capillary monolithic columns, the experimental data should be corrected for extra-column contributions. With 0.3 mm ID capillary columns, corrections for extra-column volume contributions are sufficient, but to obtain true information on the efficiency of 0.1 mm ID capillary columns, the experimental bandwidths should be corrected for extra-column contributions to peak broadening.     

Structure and performance of silica-based monolithic HPLC columns

Silica-based monolithic columns were prepared for HPLC with systematic variations of the tetramethoxysilane (TMOS) and polyethylene oxide (PEO) content as reactants in a sol-gel process accompanied by phase separation. The resulting monoliths showed differences in the macropore and silica skeleton diameter as well as in the corresponding domain sizes (the sum of macropore and skeleton diameter). All monoliths were synthesized with a diameter of 4.6 mm and cladded with a suitable polyaryletheretherketone (PEEK) polymer in a standardized and optimized manner for the subsequent chromatographic evaluation of the resulting monolithic HPLC columns. The columns were tested under normal phase conditions using n-heptane/dioxane (95:5 v/v) as a mobile phase and 2-nitroanisole as a test compound for the determination of separation efficiency and permeability. Two different sets of columns were prepared: the first one in which the amount of PEO was stepwise decreased to yield monoliths with identical macropore volumes and variations in the domain sizes. The second group of materials was synthesized adjusting both TMOS and PEO quantities to yield monolithic columns with identical macropore diameters of about 1.80 microm but different skeleton diameters and macropore volumes. The chromatographic results suggest that an increase in the column performance cannot be achieved by just arbitrarily decreasing the domain size of a given column. From a certain point of "downsizing" the monolithic structure a loss of structural homogeneity can be observed, which is apparently responsible for a lower chromatographic performance.     

Methacrylate monolithic columns of 320 microm I.D. for capillary liquid chromatography

Monolithic capillary columns (320 microm I.D.) were prepared for capillary liquid chromatography (CLC) by radical polymerization of butylmethacrylate (BMA) and ethylenedimethacrylate (EDMA) in the presence of a porogen solvent containing propan-1-ol, butane-1,4-diol and water. The influence of the contents of the porogen solvent and EDMA in the polymerization mixture on the monolith porosity and column efficiency was investigated. The composition of the polymerization mixture was optimized to attain a minimum HETP of the order of tens of microm for test compounds with various polarities. The separation performance and selectivity of the most efficient monolithic column prepared was characterized by van Deemter curves, peak asymmetry factors and Walters hydrophobicity and silanol indices. It was demonstrated that the 320-microm I.D. monolithic column exhibited CLC separation performance similar to that observed for 100- and 150-microm I.D. monolithic columns reported in the literature; moreover, the 320-microm I.D. column was easier to operate in CLC and exhibited a higher sample loadability.     

Rapid and high-resolution analysis of geometric polyprenol homologues by connected octadecylsilylated monolithic silica columns in high-performance liquid chromatography

A Chromolith Performance octadecylsilyl (ODS) monolithic silica column (Merck) was compared with a conventional microparticulate ODS-bonded silica column in the high-performance liquid chromatography separation of natural polyprenols. A system comprising two connected monolithic columns afforded an equivalent separation at half the analysis time of the conventional method. Furthermore, ten connected columns achieved a tremendously high-resolution separation, in which the complicated series of homologous polyprenols with geometric isomerism were fully separated.     

Photopolymerized organo‐silica hybrid monolithic columns: Characterization of their performance in capillary liquid chromatography

Porous hybrid organo‐silica monoliths have been prepared inside pretreated 100 μm id UV transparent fused‐silica capillaries using simultaneous sol‐gel transition and polymerization of 3‐(methacryloyloxy)propyl trimethoxysilane in the presence of toluene as a porogen. The sol‐gel reaction was catalyzed by hydrochloric acid while various photoinitiators including azobisisobutyronitrile, 2,2‐dimethoxy‐2‐phenylacetophenone, and Irgacure 819 were used for the photopolymerization carried out under irradiation with UV light at a wavelength of 254 or 365 nm. The chromatographic performance of photopolymerized monolithic columns in RP liquid chromatographic mode was assessed with respect to the following metrics: column efficiency, methylene and steric selectivity, effect of silanol groups, van Deemter plot, permeability, and pore size distribution. Columns with an efficiency of up to 77 000 plates/m for benzene has been achieved at a flow velocity of 0.47 mm/s. The performance of photopolymerized hybrid monolithic column was compared to the performance of columns prepared via thermally initiated polymerization.     

How to utilize the true performance of monolithic silica columns

Ways of utilizing the true separation efficiency of monolithic silica (MS) columns were studied. The true performance of MS columns, both regular-sized (rod-type clad with PEEK resin, 4.6 mm ID, 10 cm) and capillary sized (in 100 or 200 microm ID fused silica capillary, 25-140 cm) was evaluated by calculating the contribution of extra-column effects. HETP values of 7-9 microm were observed for solutes having retention factors (kvalues) of up to 4 for rod columns and up to 15 for a capillary column. The high permeability of MS columns allowed the use of long columns, with several connected together in the case of rod columns. Narrow-bore connectors gave good results. Peak variance caused by a column connector ranges from 50 to 70% of that caused by one rod-type column for up to three connectors or four columns in 80% methanol, but the addition of a 4th or 5th connector to add a 5th and 6th column, respectively, caused a much greater increase in peak variance, especially for long-retained solutes, which is greater than the variance caused by one rod column. Rod columns seem to show slightly lower efficiency at a pressure higher than 10 MPa or so. The use of acetonitrile-water as a mobile phase better preserved the ability of individual rod columns to generate up to 100,000 theoretical plates with 14 columns connected. Methods for eliminating extra-column effects in micro-HPLC were also studied. Split injection and on-column detection resulted in optimum performance. A long MS capillary measuring 140 cm produced 160,000 theoretical plates. The column efficiency of a capillary column was not affected by the pressure, showing advantages over the rod columns that exhibited peak broadening caused by connectors and pressure.     

Deactivation of small diameter fused silica capillary columns with organosilicon hydrides

Small diameter fused silica capillary columns (50–75 μm i.d.) were deactivated at relatively low temperatures (250–300°C) with a mixture of polymethylhydrosiloxanes and several lowmolecular-weight organosilicon hydrides. Reproducible surface deactivations of highest quality were achieved with the polymethylhydrosiloxane (PMHS) reagent. Deactivations performed with PMHS via dynamic coating with neat or diluted reagent were evaluated by gas chromatography. Deactivations achieved with organosiloxane mono-, tri-, and tetrahydrides were also evaluated and compared. Low-molecular-weight organosilicon hydride deactivations were less time consuming and required lower head pressures for filling and coating columns with the reagent. Critical surface tensions of capillary surfaces modified with PMHS and the low-molecular-weight organosilicon hydrides gave support to the dehydrocondensation reaction between silica surface silanols and silyhydride bonds of the reagents. Slopes from Zisman plots indicated that coverage of the surface ranged from highest for the polymer (PMHS) to lowest for the monomers (TMS and PMDS). Efficiency measurements showed the influence that surface modification had on the uniformity and stability of the coated capillary columns. Well-deactivated capillary columns permitted the chromatography of polar solutes using supercritical carbon dioxide as mobile phase.     

The performance of hybrid monolithic silica capillary columns prepared by changing feed ratios of tetramethoxysilane and methyltrimethoxysilane

The effect of a feed ratio of methyltrimethoxysilane (MTMS) to tetramethoxysilane (TMOS) was studied to improve the performance of a hybrid monolithic silica capillary column with 100-μm i.d. in HPLC in a range MTMS/TMOS (v/v) = 10/90–25/75. The domain size was also varied by adjusting the amount of PEG to control permeability (K = 2.8 × 10⁻¹⁴–6.9 × 10⁻¹⁴ m²). Evaluation of the performance for those capillary columns following octadecylsilylation proved an increase in retention factor (k) and a decrease in steric selectivity α(triphenylene/ortho-terphenyl) with the increase in MTMS content in the feed. The effect of the feed ratio was also observed in porosity and hydrophobic property of the C18 stationary phase from the results of size exclusion chromatography (SEC) and reversed phase characterization. The monolithic silica capillary columns prepared under new preparation conditions were able to produce a plate height of 4.6–6.0 μm for hexylbenzene in a mobile phase acetonitrile/water = 80/20 at a linear velocity of 2 mm/s. Consequently, it was possible to prepare hybrid monolithic silica capillary columns with higher performance than those reported previously while maintaining the retention factors in a similar range by reducing the MTMS/TMOS ratio and increasing the total silane concentration in feed.     

Influence of the diameter of monolithic capillary columns on their gas chromatography characteristics

Divinylbenzene polymer monolithic capillary columns were prepared on the basis of capillaries 0.01 to 0.53 mm in diameter. Separation properties of the columns were investigated with the use of a test mixture of light hydrocarbons. The permeability and C parameter in the Van Deemter equation were determined for all the columns. For the most part, the columns had similar characteristics: permeability was in the range (2.2 ± 0.2) × 10^?9 cm², with parameter C in the range (0.7 ± 0.2) × 10^?3 s (with n-butane as a sorbate). It was thus established that capillary diameter has only a slight effect on the efficiency of monolithic capillary columns (unlike packed capillary columns and microcolumns, whose properties, according to the literature data, depend strongly on the column diameter). The difference in properties between the narrowest monolithic column (capillary diameter 0.01 mm) and the others is explained by column overloading.     

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.     

Retention characteristics of aromatic hydrocarbons on silica and aminopropyl-modified monolithic columns in normal-phase HPLC

A suite of aromatic hydrocarbons were used to investigate the influence of structure upon retention in normal-phase HPLC mode using bare silica and aminopropyl-modified silica Chromolith type monolithic columns. The aminopropyl-modified silica monolith was used to separate a complex petroleum fraction based on double bond equivalence.     

Characterization of monolithic columns for HPLC

Monolithic stationary phases and columns have rapidly become highly popular separation media for liquid chromatography, in spite of their recent discovery. However, their most important features have not yet been completely clarified. A complete understanding of their performance and of their intrinsic characteristics will require the systematic acquisition of many series of reliable experimental data and their consistent analysis from different points of view. Progress in their design and production requires now that the chromatographic behavior of monolithic columns be studied in close connection with their physico-chemical and structural properties. The main goal of this review is to summarize fundamental information on some physico-chemical and chromatographic characteristics of monolithic stationary phases and columns for RPLC. The material reviewed deals only with silica-based monolithic columns. First, structural information on the porosities and the size of the pores in monolithic columns is reported. Second, results of chromatographic studies that deal with the characterization of monolithic columns are summarized. Third, results of detailed studies made on the adsorption equilibrium and the surface heterogeneity of monolithic stationary phases are presented. Finally, results on the mass transfer kinetics in monolithic columns derived from the applications of the classical random-walk model and of the moment theory to a new model of the monolith are discussed.     

A study of the efficiency of monolithic silica gel capillary columns for gas chromatography

The efficiency and dynamic characteristics of seven silica-gel-based monolithic capillary columns were analyzed by separating on them a mixture of five light hydrocarbons. For helium carrier gas flowing at an optimum velocity, the height equivalent to a theoretical plate was found to be 0.15–0.20 mm, values comparable to those typical of packed capillary columns. An analysis of the Van Deemter curves for the columns under study demonstrated that the main contribution to the smearing of the chromatographic zone comes from the diffusional processes in the mobile phase while the mass transfer between the mobile and stationary phases plays only a minor role. At the same time, the parameter A in the Van Deemter equation, which characterizes the degree of column packing uniformity, was found to be negative. This result contradicts the classical theory of chromatography and calls for further studies of monolithic capillary columns.     

Applications of silica-based monolithic HPLC columns

The recent invention and successive commercial introduction of monolithic silica columns has motivated many scientists from both academia and industry to study their use in HPLC. The first paper on monolithic silica columns appeared in 1996. Currently about 200 papers have been published relating to applications and characterization of monolithic silica columns, including monolithic capillaries. This review attempts to give an overview covering various aspects of this new column type in the field of high throughput analysis of drugs and metabolites, chiral separations, analysis of pollutants and food-relevant compounds, as well as in bioanalytical separations such as in proteomics. Some of the applications are described in greater detail. The numerous publications dealing with the physicochemical and chromatographic characterization of monolithic silica columns are briefly summarized.     

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.     

HILIC mode separation of polar compounds by monolithic silica capillary columns coated with polyacrylamide

HILIC mode columns were prepared by an on-column polymerization of acrylamide on a monolithic silica capillary column modified with N-(3-trimethoxysilylpropyl)methacrylamide as the anchor group. The products showed HILIC mode retention characteristics with three times greater permeability and slightly higher column efficiency compared to a commercially available amide-type HILIC column packed with 5-μm particles. The selectivity of the monolithic silica-based column was similar to that of the particulate column for each group of solutes towards nucleosides, nucleic bases and carbohydrate derivatives, although a considerable difference was observed in the selectivity for the solute groups. Although the retention of solutes based on the polar functionality was much smaller with the monolithic silica columns, which had a smaller phase ratio, than with the particle-packed column, the former can achieve better separation utilizing the high permeability and higher column efficiencies of a longer column.     

Performance evaluation of long monolithic silica capillary columns in gradient liquid chromatography using peptide mixtures

A systematic study is reported on the performance of long monolithic capillary columns in gradient mode. Using a commercial nano-LC system, reversed-phase peptide separations obtained through UV-detection were conducted. The chromatographic performance, in terms of conditional peak capacity and peak productivity, was investigated for different gradient times (varying between 90 and 1320min) and different column lengths (0.25, 1, 2 and 4m) all originating from a single 4m long column. Peak capacities reaching values up to n=10(3) were measured in case of the 4m long column demonstrating the high potential of these long monoliths for the analysis of complex biological mixtures, amongst others. In addition, it was found that the different column fragments displayed similar flow resistance as well as consistent chromatographic performance in accordance with chromatographic theory indicating that the chromatographic bed of the original 4m long column possessed a structural homogeneity over its entire length.