Improved endcapping method of monolithic silica columns

Following a bonding reaction with octadecyldimethyl-N,N-diethylaminosilane (ODS-DEA), a monolithic silica was subjected to consequential on-line endcapping reactions with hexamethyldisilazane (HMDS) or N-(trimethylsilyl)imidazole (TMSI) to compare the two reagents. Endcapping of monolithic silica C(18) columns prepared by a manufacturer was also studied. After endcapping with TMSI in acetonitrile (ACN), all columns provided better performance for hydrogen-bonding compounds and basic compounds than C(18) phases endcapped with HMDS. TMSI endcapping of commercial Chromolith Performance columns also gave improved performance. The results indicate that one can significantly improve the performance of monolithic silica C(18) columns for basic compounds by a simple on-column reaction with TMSI in ACN at a relative low temperature.     

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.     

A strategy to develop fast RP-HPLC methods using monolithic silica columns

Since the appearance of monolithic silica, much work has been done describing the properties of monolithic silica columns. Meanwhile the transferability of analytical methods from conventional to monolithic silica columns has been intensively investigated [1-5]. RP HPLC method development strategies for conventional columns should be updated or scaled to meet the higher performing monolithic column technology. Because of the high permeability of monolithic silica columns it should be possible to decrease the time for method development by applying high isocratic flow rates. Here we suggest a clear strategy for method development using monolithic columns. The strategy will be applicable for various sample compositions, e. g., acidic, basic, or neutral. The applicability of monolithic columns for especially complex separations of basic mixtures without the need of using a highly basic mobile phase that harms the column will be pointed out in this work. This work will describe in detail the actual method development process. For better understanding of our strategy, the influence of flow rate, column length, mobile phase composition, pH, and temperature will be discussed. Details about the application of a flow program will be mentioned.     

Preparation of monolithic silica columns for high-performance liquid chromatography

Preparation methods of monolithic silica columns for HPLC including the surface modification were reviewed. Chemical modification methods recently reported to obtain stationary phases for reversed-phase (RP), chiral, ion-exchange, and hydrophilic interaction chromatography (HILIC) separations were discussed. Recent results related to preparation methods of monolithic silica were also covered. The characteristics and properties of silica monoliths and some applications of monolithic silica columns for different analytical and bioanalytical fields will be commented.     

A computational study of the porosity effects in silica monolithic columns

We report on a theoretical study of the influence of the through-pore porosity on the main chromatographic performance parameters (reduced theoretical plate height, flow resistance, and separation impedance) of silica monoliths. To investigate this problem devoid of any structural uncertainties, computer-generated structural mimics of the pore geometry of silica monolithic columns have been studied. The band broadening in these synthetic monoliths was determined using a commercial Computational Fluid Dynamics (CFD) software package. Three widely differing external porosities (epsilon = 0.38, epsilon = 0.60, and epsilon = 0.86) are considered and are compared on the basis of an identical intra-skeleton diffusivity (Ds = 5 x 10(-10)m2/s), internal porosity (epsilon(int) = 0.5), and for the same phase retention factor (k' = 1.25). Since the data are obtained for perfectly ordered structures, the calculated plate heights and separation impedances constitute the ultimate performance ever to be expected from a monolithic column. It is found that, if silica monoliths could be made perfectly homogeneous, domain size-based reduced plate heights as small as h(min) approximately 0.8 (roughly independent of the porosity) and separation impedances as small as Emin approximately 130 (epsilon = 0.60) and Emin approximately 40 (epsilon = 0.86) should be achievable with pure water as the working fluid. The data also show that, although the domain size is a much better reduction basis than the skeleton size, the former is still not capable of bringing the van Deemter curves of different porosity columns into perfect agreement in the C term dominated velocity range. It is found that, in this range, large porosity monoliths can be expected to yield smaller domain size-based reduced plate heights than small porosity monoliths.     

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.     

Short communication performance of octadecylsilylated monolithic silica capillary columns of 530 microm inner diameter in HPLC

Monolithic silica capillary columns were successfully prepared in a fused silica capillary of 530 microm inner diameter and evaluated in HPLC after octadecylsilylation (ODS). Their efficiency and permeability were compared with those of columns pakked with 5-microm and 3-microm ODS-silica particles. The monolithic silica columns having different domain sizes (combined size of through-pore and skeleton) showed 2.5-4.0-times higher permeability (K= 5.2-8.4 x 10(-14) m2) than capillary columns packed with 3-mm particles, while giving similar column efficiency. The monolithic silica capillary columns gave a plate height of about 11-13 microm, or 11 200-13 400 theoretical plates/150 mm column length, in 80% methanol at a linear mobile phase velocity of 1.0 mm/s. The monolithic column having a smaller domain size showed higher column efficiency and higher pressure drop, although the monolithic column with a larger domain size showed better overall column performance, or smaller separation impedance (E value). The larger-diameter (530 microm id) monolithic silica capillary column afforded a good peak shape in gradient elution of proteins at a flow rate of up to 100 microL/min and an injection volume of up to 10 microL.     

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.     

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.     

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.     

Pore structural characterization of monolithic silica columns by inverse size-exclusion chromatography

In this work, a parallel pore model (PPM) and a pore network model (PNM) are developed to provide a state-of-art method for the calculation of several characteristic pore structural parameters from inverse size-exclusion chromatography (ISEC) experiments. The proposed PPM and PNM could be applicable to both monoliths and columns packed with porous particles. The PPM and PNM proposed in this work are able to predict the existence of the second inflection point in the experimental exclusion curve that has been observed for monolithic materials by accounting for volume partitioning of the polymer standards in the macropores of the column. The appearance and prominence of the second inflection point in the exclusion curve is determined to depend strongly on the void fraction of the macropores (flow-through pores), (b) the nominal diameter of the macropores, and (c) the radius of gyration of the largest polymer standard employed in the determination of the experimental ISEC exclusion curve. The conditions that dictate the appearance and prominence of the second inflection point in the exclusion curve are presented. The proposed models are applied to experimentally measured ISEC exclusion curves of six silica monoliths having different macropore and mesopore diameters. The PPM and PNM proposed in this work are able to determine the void fractions of the macropores and silica skeleton, the pore connectivity of the mesopores, as well as the pore number distribution (PND) and pore volume distribution (PVD) of the mesopores. The results indicate that the mesoporous structure of all materials studied is well connected as evidenced by the similarities between the PVDs calculated with the PPM and the PNM, and by the high pore connectivity values obtained from the PNM. Due to the fact that the proposed models can predict the existence of the second inflection point in the exclusion curves, the proposed models could be more applicable than other models for ISEC characterization of chromatographic columns with small diameter macropores (interstitial pores) and/or large macropore (interstitial pore) void fractions. It should be noted that the PNM can always be applied without the use of the PPM, since the PPM is an idealization that considers an infinitely connected porous medium and for materials having a low (<6) pore connectivity the PPM would force the PVD to a lower average diameter and larger distribution width as opposed to properly accounting for the network effects present in the real porous medium.     

High efficiency, high temperature separations on silica based monolithic columns

The effect of temperature on separation using reversed-phase monolithic columns has been investigated using a nano-LC pumping system for gradient separation of tryptic peptides with MS detection. A goal of this study was to find optimal conditions for high-speed separations. The chromatographic performance of the columns was evaluated by peak capacity and peak capacity per time unit. Column lengths ranging from 20 to 100 cm and intermediate gradient times from 10 to 30 min were investigated to assess the potential of these columns in a final step separation, e.g. after fractionation or specific sample preparation. Flow rates from 250 to 2000 nL/min and temperatures from 20 to 120°C were investigated. Temperature had a significant effect on fast separations, and a flow rate of 2000 nL/min and a temperature of 80°C gave the highest peak capacity per time unit. These settings produced 70% more protein identifications in a biological sample compared to a conventional packed column. Alternatively, an equal amount of protein identifications was obtained with a 40% reduction in run time compared to the conventional packed column.     

Use of gel-casting to prepare HPLC monolithic silica columns with uniform mesopores and tunable macrochannels

Monolithic silica columns with defined mesopore and a tunable macropore structure were fabricated by the gel-casting of ordered mesoporous silica precursors.     

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.     

Impact of pore structural parameters on column performance and resolution of reversed-phase monolithic silica columns for peptides and proteins

In this work, monolithic silica columns with the C4, C8, and C18 chemistry and having various macropore diameters and two different mesopore diameters are studied to access the differences in the column efficiency under isocratic elution conditions and the resolution of selected peptide pairs under reversed-phase gradient elution conditions for the separation of peptides and proteins. The columns with the pore structural characteristics that provided the most efficient separations are then employed to optimize the conditions of a gradient separation of a model mixture of peptides and proteins based on surface chemistry, gradient time, volumetric flow rate, and acetonitrile concentration. Both the mesopore and macropore diameters of the monolithic column are decisive for the column efficiency. As the diameter of the through-pores decreases, the column efficiency increases. The large set of mesopores studied with a nominal diameter of approximately 25 nm provided the most efficient column performance. The efficiency of the monolithic silica columns increase with decreasing n-alkyl chain length in the sequence of C18相似文献   

Capillary electrochromatography with monolithic silica columns. V. Study of the electrochromatographic behaviors of polar compounds on monolithic silica having surface bound cyano functionalities

In this report, a novel polar monolithic capillary column is described for normal phase CEC (NP-CEC) of representative polar compounds including mono- and oligosaccharides, peptides, and basic drugs. The polar monolithic column, which was described in detail in the preceding paper, consisted of silica-based monolith bonded with 1H-imidazole-4,5-dicarbonitrile (IDCN) and is denoted as 2CN-OH-Monolith. Various retention parameters for neutral polar solutes (e.g., mono- and oligosaccharides) and charged polar solutes (e.g., peptides and basic drugs) were evaluated over a wide-range of elution conditions. These retention parameters yielded quantitative assessment for the polar interactions between the model solutes and the stationary phase under investigation as well as the effect of electromigration of charged solutes on their overall migration in NP-CEC. Furthermore, this investigation demonstrated that despite the possibility of achieving isocratic separation in NP-CEC for widely differing polar species, multistep-gradient elution in NP-CEC is preferred to bring about the rapid separation of a large number of polar species in a single run.     

High-performance liquid chromatographic enantioseparations on capillary columns containing crosslinked polysaccharide phenylcarbamate derivatives attached to monolithic silica

Monolithic capillary columns containing native silica gel were covalently modified with 3,5-disubstituted phenylcarbamate derivatives of cellulose and amylose and applied for enantioseparations in capillary LC. The method previously used for covalent immobilization of polysaccharide phenylcarbamate derivatives onto the surface of microparticulate silica gel was successfully adapted for in situ modification of monolithic fused-silica capillary columns. The effects of the nature of polysaccharide and the substituents, as well as of multiple covalent immobilization of polysaccharide derivative on chromatographic performance of capillary columns were studied. The capillary columns obtained using this technique are stable in all solvents commonly used in LC and exhibit promising enantiomer resolving ability.     

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.     

Effect of the nature of the carrier gas on the chromatographic characteristics of monolithic silica capillary columns

Chromatographing a model mixture of hydrocarbons with various carrier gases (helium, hydrogen, nitrogen, carbon dioxide, and nitrous oxide) was used to study the separation ability of monolithic silica capillary columns. It was revealed that the nature of the carrier gas strongly affects the retention time of the sorbates and the height equivalent to a theoretical plate (HETP) of the column, with the values of both these parameters decreasing in the series He > H₂ > N₂ > CO₂ ～ N₂O. This effect was found to be more pronounced for normal hydrocarbons than for their isomers. For chromatographing with CO₂ or N₂O under optimum conditions, the HETP was 25–30 μm, a value indicative of a higher specific efficiency of monolithic capillary columns. Theoretical correlations between the HETP and the properties of the mobile phase were considered. As a result, it was concluded that elevated pressures of the carrier gas, which are required to ensure the optimum operation of monolithic capillary columns, may affect the properties of the chromatographic system.