Monolithic Silica Column Research Articles

Single-step approach for fabrication of vancomycin-bonded silica monolith as chiral stationary phase

A vancomycin-bonded silica monolithic column for capillary electrochromatography (CEC) was prepared by a single-step in situ sol–gel approach. This sol–gel process incorporates a synthetic sol–gel precursor which contains a macrocyclic antibiotic, vancomycin, to form a porous silica network inside a fused-silica capillary. To avoid degradation of vancomycin during the column fabrication, a mild step was adopted into the sol–gel process. The performance of the vancomycin chiral stationary phase was investigated by CEC in both the reversed-phase mode and the normal-phase mode. The vancomycin chiral stationary phase was optimized with respect to vancomycin loading in the reversed-phase mode for chiral separation of thalidomide enantiomers. The best efficiency and resolution values of 94600plates/m and 5.79, respectively, were achieved. The optimized column was further applied to chiral separation of alprenolol enantiomers. A plate height of less than 7μm for the first eluted enantiomer of alprenolol was obtained in an aqueous mobile phase at a flow rate of 0.74mm/s. Using enantiomers of seven β-blockers and some other basic enantiomers as test analytes, separation efficiencies of up to 148100plates/m in the reversed-phase mode and up to 138100plates/m in the normal-phase mode were achieved.

One-pot synthesis of a monolithic silica capillary column bed for μ-HPLC using a template induced method

In this study we report a new, simple and efficient method for the synthesis of a silica monolithic chromatography column. Instead of the usual base corrosion method, a template induced synthesis method was used to create bimodal pores in the column bed. Block polymer Pluronic F127 was used as a dual-function template to form a hierarchically porous silica monolith with both macropores and mesopores. The surface of the silica monolithic column bed was modified in situ by the C18 reagent, and was then evaluated by the μ-HPLC method. 30 000–40 000 theoretical plates per m were observed indicating that the new synthesis method creates a monolithic material that can act as an efficient chromatographically stationary phase. The method also makes the fabrication of silica monolithic columns (especially capillary columns) much simpler.

Simultaneous determination of N(G)-monomethyl-L-arginine, N(G),N(G)-dimethyl-L-arginine, N(G),N(G')-dimethyl-L-arginine, and L-arginine using monolithic silica disk-packed spin columns and a monolithic silica column.

We have developed and validated a high-performance liquid chromatography method that uses monolithic silica disk-packed spin columns and a monolithic silica column for the simultaneous determination of N(G)-monomethyl-L-arginine, N(G),N(G)-dimethyl-L-arginine, and N(G),N(G')-dimethyl-L-arginine in human plasma. For solid-phase extraction, our method employs a centrifugal spin column packed with monolithic silica bonded to propyl benzenesulfonic acid as a cation exchanger. After pretreatment, the methylated arginines are converted to fluorescent derivatives with 4-fluoro-7-nitro-2,1,3-benzoxadiazole, and then the derivatives are separated on a monolithic silica column. L-arginine concentration was also determined in diluted samples. Standard calibration curves revealed that the assay was linear in the concentration range 0.2-1.0 μM for methylated arginines and 40-200 μM for L-arginine. Linear regression of the calibration curve yielded equations with correlation coefficients of 0.999 (r(2)). The sensitivity was satisfactory, with a limit of detection ranging from 3.75 to 9.0 fmol for all four compounds. The RSDs were 4.3-4.8% (intraday) and 3.0-6.8% (interday). When this method was applied to samples from six healthy donors, the detected concentrations of N(G)-monomethyl-L-arginine, N(G),N(G)-dimethyl-L-arginine, N(G),N(G')-dimethyl-L-arginine and L-arginine were 0.05 ± 0.01, 0.41 ± 0.07, 0.59 ± 0.11, and 83.8 ± 30.43 μM (n = 6), respectively.

Expanding the separation capability of sequential injection chromatography: Determination of melamine in milk exploiting micellar medium and on-line sample preparation

Silica, C8 and C18 monolithic column were the only option for separation on a sequential injection chromatography (SIC) system due its low-pressure operation. Therewith, the separation of molecules poorly retained by this stationary phase was a difficult task. This work presents the first use of micellar chromatography on a SIC system, with the applicability demonstrated by the determination of melamine in milk. The mobile phase was composed by aqueous sodium dodecyl sulfate (SDS) solution and propanol (92.5:7.5). The adsorption of SDS monomers on the C18 stationary phase modified its chromatographic properties, enabling satisfactory melamine separation from the milk components. The sample pretreatment procedure was on-line implemented by dilution of the sample with SDS using a multiposition valve. Calibration equations obtained from melamine solutions prepared in different types of milk and water presented similar sensitivity, indicating absence of matrix effects. A linear response was observed within 2.0 and 6.0mgL−1 of melamine with a detection limit estimated at 0.6mgL−1 and coefficients of variation at 2.9% (n=6). The procedure was suitable for fast determination of melamine below the limits established by FDA and WHO (2.5mgL−1), with minimized reagent consumption. Results for different milk samples agreed with those obtained by high performance liquid chromatography at the 95% confidence level.

Preparation of a high specific surface area monolithic silica reversed phase chromatography column using a template induced method

In this study we report a new method for the synthesis of a silica monolithic reverse phase chromatography column. Instead of the usual base corrosion method a template induced synthesis method was used to create bimodal pores in the column bed. Block polymer Pluronic F127 was used as a dual-function template to form a hierarchically porous silica monolith with both macropores and mesopores. Poly(ethylene glycol) was used in combination with F127 to effectively control the mesopore size distribution and the volume shrinkage during the gelation step. The resultant material showed a much narrower mesopore size distribution (centered at 6 nm) than that using F127 as the only template reagent. The surface of the silica monolithic column bed was modified in situ by the C18 reagent, and was then evaluated by a μ-HPLC method. A large number of theoretical plates were observed (>110 000/m) indicating that the new synthesis method creates a monolithic material that can act as a chromatographically efficient stationary phase. The method also makes the fabrication of the silica monolithic column (especially capillary columns) much simpler.

“Thiol-ene” photoclick chemistry as a rapid and localizable functionalization pathway for silica capillary monolithic columns

Herein, we report the “ene-thiol” photografting of 1-octadecanethiol onto vinyl pre-functionalized silica monolith to prepare clicked reversed-phase silica monolithic columns with high permeability and performances. The experimental conditions (concentration of thiol in solution, irradiation duration) are optimized with respect to highest retention and methylene selectivity, i.e. to the highest surface coverage of the monolith. It is demonstrated that an irradiation duration of 5min is enough with a 0.8M concentration of 1-octadecanethiol in solution or that it may be replaced by successive irradiations at a lower ODT concentration (0.19M) with renewing of the solution between the illuminations. Retention factors as high as those obtained with standard silanization are reached while keeping the intrinsic monolith permeability and efficiency (160,000plates/m in nano-LC at 0.7mm/s). The absence of polymerization, in the “ene-thiol” version, is demonstrated. Indeed, the steric selectivity of our clicked-material is characteristic of monolayer-like functionalized silica and significantly lower than the steric selectivity measured on polymeric-like functionalized silica.

Assessing the performance of curtain flow first generation silica monoliths

Analytical scale active flow technology first generation silica monolithic columns kitted out in curtain flow mode of operation were studied for the first time. A series of tests were undertaken assessing the column efficiency, peak asymmetry and detection sensitivity. Two curtain flow columns were tested, one with a fixed outlet ratio of 10% through the central exit port, the other with 30%. Tests were carried out using a wide range in inlet flow segmentation ratios. The performance of the curtain flow columns were compared to a conventional monolithic column. The gain in theoretical plates achieved in the curtain flow mode of operation was as much as 130%, with almost Gaussian bands being obtained. Detection sensitivity increased by as much as 250% under optimal detection conditions. The permeability advantage of the monolithic structure together with the active flow technology makes it a priceless tool for high throughput, sensitive, low detection volume analyses.

Experimental and numerical validation of the effective medium theory for the B-term band broadening in 1st and 2nd generation monolithic silica columns

Effective medium theory (EMT) expressions for the B-term band broadening in monolithic silica columns are presented at the whole-column as well as at the mesoporous skeleton level. Given the bi-continuous nature of the monolithic medium, regular as well as inverse formulations of the EMT-expressions have been established. The established expressions were validated by applying them to a set of experimental effective diffusion (Deff)-data obtained via peak parking on a number of 1st and 2nd generation monolithic silica columns, as well as to a set of numerical diffusion simulations in a simplified monolithic column representation (tetrahedral skeleton model) with different external porosities and internal diffusion coefficients. The numerically simulated diffusion data can be very closely represented over a very broad range of zone retention factors (up to k″=80) using the established EMT-expressions, especially when using the inverse variant. The expressions also allow representing the experimentally measured effective diffusion data very closely. The measured Deff/Dmol-values were found to decrease significantly with increasing retention factor, in general going from about Deff/Dmol=0.55 to 0.65 at low k″ (k″≅1.5–3.8) to Deff/Dmol=0.25 at very high k″ (k″≅40–80). These values are significantly larger than observed in fully-porous and core–shell particles. The intra-skeleton diffusion coefficient (Dpz) was typically found to be of the order of Dpz/Dmol=0.4, compared to Dpz/Dmol=0.2–0.35 observed in most particle-based columns. These higher Dpz/Dmol values are the cause of the higher Deff/Dmol values observed. In addition, it also appears that the higher internal diffusion is linked to the higher porosity of the mesoporous skeleton that has a relatively open structure with relatively wide pores. The observed (weak) relation between Dpz/Dmol and the zone retention factor appears to be in good agreement with that predicted when applying the regular variant of the EMT-expression directly to the mesoporous skeleton level.

A novel dilute and shoot HPLC assay method for quantification of irbesartan and hydrochlorothiazide in combination tablets and urine using second generation C18-bonded monolithic silica column with double gradient elution

Irbesartan (IRB) and hydrochlorothiazide (HCT) are angiotensin-II receptor antagonist and thiazide-class diuretic compounds, respectively, which are in use in the treatment of hypertension. A novel dilute-and-shoot HPLC assay method for simultaneous quantification of IRB and HCT in fixed-dose combination tablets and urine samples was described. The separation of IRB, HCT and agomelatine (internal standard) was carried out using a second generation C18-bonded monolithic silica column (Chromolith(®) High Resolution RP-18e, 100×4.6mm, Merck KGaA), utilizing both mobile phase and flow rate gradient elution programs. The analytes were detected at 230 nm wavelength using photodiode array detector within 24 minutes with high resolution, observing about 50 percent more peak capacity when using second generation C18-bonded monolithic silica column. Urine samples were introduced into the system effortlessly, with only filtration and subsequent dilution. Validation studies were performed according to the official recommendations of USP and ICH, and the developed method was successfully applied to pharmaceutical tablets and urine samples.

Preparation of nanoparticles-modified silica monolith for on-column surface enhanced Raman spectroscopy

A novel silica monolith modified with Ag/Au nanoparticles was prepared for the on-column surface enhanced Raman spectroscopy (SERS). The bare monolithic silica column was prepared from in-situ co-condensation of tetraethoxysilane (TMOS) and methyl trimethoxysilane (MTMS) in the presence of polyethylene glycol (PEG) via a sol-gel process in the capillary, and was chemically modified with (3-mercaptopropyl) trimethoxysilane (MTPMS), followed by immobilization of Ag/Au nanoparticles. Transmission electron microscopy (TEM) and UV-Vis spectrometer were used to collect the TEM images and the extinction spectra of the nanoparticles colloid, respectively. Scanning electron microscope (SEM) was utilized to record the morphology of the silica monolith The authros used p-aminothiophenol (PATP) as a probing molecule, and the SERS effect was investigated on Au/Ag nanoparticle-modified silica monolith under the excitation line of 633 and 532 nm, respectively. It is concluded that nanoparticle-modified silica monoliths will have broad application to the on-site detection of food and water contaminants in the field.

Hydrophilic Interaction Chromatography Using a Meter-Scale Monolithic Silica Capillary Column for Proteomics LC-MS

A meter-scale monolithic silica capillary column modified with urea-functional groups for hydrophilic interaction liquid chromatography (HILIC) was developed for highly efficient separation of biological compounds. We prepared a ureidopropylsilylated monolithic silica capillary column with a minimum plate height of 12 μm for nucleosides and a permeability of 2.1 × 10(-13) m(2), which is comparable with the parameters of monolithic silica-C18 capillary columns. Over 300,000 theoretical plates were experimentally obtained in HILIC with a 4 m long column at 8 MPa; this is the best result yet reported for HILIC. A 2 m long ureidopropylsilylated monolithic silica capillary column was utilized to develop a HILIC mode LC-MS system for proteomics applications. Using tryptic peptides from human HeLa cell lysate proteins, we identified the comparable numbers of peptides and proteins in HILIC with those in reversed-phase liquid chromatography (RPLC) using a C18-modified monolithic silica column when shallow gradients were applied. In addition, approximately 5-fold increase in the peak response on average was observed in HILIC for commonly identified tryptic peptides due to the high acetonitrile concentration in the HILIC mobile phase. Since HILIC mode LC-MS shows orthogonal selectivity to RPLC mode LC-MS, it is useful as a complementary tool to increase proteome coverage in proteomics studies.

An automated materials screening approach for the development of sol–gel derived monolithic silica enzyme reactor columns

Fabrication of monolithic protein-doped capillary columns was reported almost 10 years ago. These columns were derived from a diglycerylsilane precursor, however this material is not commercially available, is difficult to produce in large quantities and has very short phase separation and gelation times, which leads to issues with column reproducibility. Herein, we investigate the use of sodium silicate (SS), a commercially available biofriendly sol–gel precursor, for the fabrication of bimodal meso/macroporous protein-doped monolithic silica columns that are suitable for immobilized enzyme reactor (IMER) assays. Using an automated liquid handler and platereader, a hierarchical materials screening approach was applied to ∼1400 formulations, from which we have identified materials with long gelation times that can form robust bimodal meso/macroporous materials suitable for fabrication of monolithic silica columns. A subset of these materials was observed to have good chromatographic behavior (appropriate backpressure and good stability). A secondary screen around lead materials was performed to identify optimal materials for fabrication of IMER columns. These materials were tested for leaching and activity of immobilized acetylcholine esterase to identify an optimal material for IMER column fabrication. The optimal material was formed from 2% (w/v) silica which was combined with 1.25% PEG 600 at pH 6.4 in 100 mM TRIS buffer. Such columns showed reproducible IMER performance and were able to quantitatively measure the inhibition of immobilized AChE by galanthamine with an inhibition constant of 175 ± 5 nM, which is in excellent agreement with the literature value.

Band broadening along gradient reversed phase columns: A potential gain in resolution factor

The advantages of using columns packed with stationary phases having a composition gradient so that retention factors increase toward the outlet and eluting them with an isocratic mobile phase may provide increased peak resolution in liquid chromatography. This approach is discussed from a theoretical viewpoint. The retention factor is assumed to increase linearly along the column. The peak width can be predicted under three different conditions: (1) the possible band compression is neglected (the Giddings model); (2) band compression is taken into account (the Poppe model); and (3) both band compression and extra-column effects are considered in the calculations. The impact of a stationary phase gradient on the resolution of a pair of compound difficult to separate (constant selectivity α=1.05) is illustrated for a 3mm×100mm silica monolithic column of the second generation (porosity ϵt=0.85, plate height H=6μm) operated with a new generation of liquid chromatograph (pre- and post-column volume variances of 10 and 5μL2, respectively). The results show that the resolution factor RS becomes optimum for a specific positive value of the retention gradient along the column. This optimum depends on the retention factor at the column inlet.

Regeneration of a silica monolithic rod column using harsh methods followed by firm thermodynamic and kinetic validation

In this study, a numerical tool is introduced--based on thermodynamic and kinetic separation theory--for validating the regeneration of monolithic rod columns after cutting their inlet sections. A long-used RP-18e monolithic column was deemed to be unfit for further coffee analysis because of poor separation performance. The columns brownish inlet section was physically removed with a lathe, leaving a clean white inlet section. The original and regenerated columns were extensively analyzed and compared using numerical tools for processing adsorption data. The perturbation peak method was used to measure the adsorption isotherm of phenol on the original and regenerated monolith and the adsorption energy distributions were calculated for identifying any change in the degree of heterogeneity. Although peak shapes improved considerably after regeneration, no significant differences were found in the detailed characterization of the processed adsorption data between the original column and the regenerated one. This indicates that the removal of a section of the monolithic bed can be undertaken without damaging the column and columns in which their inlet head sections are contaminated may still function with normal adsorption behavior. In addition, the combined thermodynamic and kinetic methodology could accurately be used to evaluate any regeneration method of columns.

Phospholipid-Modified ODS Monolithic Column for Affinity Prediction of Hydrophobic Basic Drugs to Phospholipids

A phospholipid-modified octadecyl silica (ODS) monolithic column was prepared and its interaction with basic hydrophobic drugs was studied. These drugs are of interest in pharmaceutical research because of their strong interaction with biomembranes. The amount of dimyristoylphosphatidylcholine trapped on the ODS surface was reproducible, and the results of the trinitrobenzenesulfonic acid assay suggested the formation of a monolayer on the surface. Both hydrophobic and electrostatic interactions acted between the model drugs and the phosphatidylcholine. The column was stable for 10 days at least. The column was applied to the affinity screening of basic drugs to phospholipid. Good correlation was obtained between log k and log P for the basic drugs lidocaine, quinidine, propranolol, imipramine, and chlorpromazine. The monolithic silica column allowed highly hydrophobic basic drugs such as imipramine and chlorpromazine to be assayed; these are difficult to analyze by using a conventional particle-packed column. These drugs were clearly separated from acidic drugs naproxen and warfarin on the log k versus log P plots. The thermodynamic studies revealed that the retention of the drug was an enthalpy-driven process, and that the decrease in enthalpy for the phospholipid-modified ODS monolithic column was larger than those for immobilized artificial membrane columns. Our results suggest that the phospholipid-modified ODS monolithic column is applicable to affinity screening of drugs to phospholipids.

Simultaneous analysis of nanoparticles and small molecules by high-performance liquid chromatography using a silica monolithic column.

A high-performance liquid chromatography method using a commercially available silica monolithic column for the simultaneous analysis of nanoparticles and small molecules was developed. The method uses the micrometer-sized flow-through pores and nanometer-sized mesopores of the monolithic column for separation: first, size separation of nanoparticles was performed by the micrometer-sized pores using the hydrodynamic mode, and then small molecules were separated by the nanometer-sized pores using the normal-phase mode. The method was used to evaluate and compare three existing methods for purifying nanoparticles and to analyse nanoparticle stability. The bimodal structure of the monolithic column is promising for the simultaneous separation of nanoparticles and small molecules.

Photopolymerization of acrylamide as a new functionalization way of silica monoliths for hydrophilic interaction chromatography and coated silica capillaries for capillary electrophoresis

A simple, rapid and localizable photochemical process for the preparation of hydrophilic coated capillary and silica-based monolithic capillary columns is described. The process involves the free radical polymerization of acrylamide monomers onto acrylate pre-activated silica surface triggered by UV photoinitiation. The experimental conditions (monomer content, time of irradiation) were optimized on silica monolithic columns by monitoring the evolution of the chromatographic properties (retention, permeability, efficiency) in HILIC mode using a set of nucleosides as test solutes. Compared to thermal polymerization process, the photoinitiation allows the preparation of highly retentive and efficient HILIC monolithic columns in less than 10min of irradiation. This process was then successfully applied to the surface coating of fused silica capillary walls. In addition to its relative high stability and ability to reduce the electroosmotic flow, this polyacrylamide coating is localizable. Benefits of this localizable photochemical process are highlighted through the conception of an in-line integrated bimodal microseparation tool combining a SPE preconcentration step on a photografted silica monolith and an electrokinetic separation step in a polyacrylamide photopolymerized capillary section. Two neuropeptides are used as model solutes to illustrate the suitability of this approach.

Detailed characterization of the kinetic performance of first and second generation silica monolithic columns for reversed-phase chromatography separations

The kinetic performance of commercially available first generation and prototype second generation silica monoliths has been investigated for 2.0mm and 3.0–3.2mm inner diameter columns. It is demonstrated that the altered sol–gel process employed for the production of second generation monoliths results in structures with a smaller characteristic size leading to an improved peak shape and higher efficiencies. The permeability of the columns however, decreases significantly due to the smaller throughpore and skeleton sizes. Scanning electron microscopy pictures suggest the first generation monoliths have cylindrical skeleton branches, whereas the second generation monoliths rather have skeleton branches that resemble a single chain of spherical globules. Using recently established correlations for the flow resistance of cylindrical and globule chain type monolithic structures, it is demonstrated that the higher flow resistance of the second generation monoliths can be entirely attributed to their smaller skeleton sizes, which is also evident from the external porosity that is largely the same for both monolith generations (ɛe∼0.65). The recorded van Deemter plots show a clear improvement in efficiency for the second generation monoliths (minimal plate heights of 13.6–14.1μm for the first and 6.5–8.2μm for the second generation, when assessing the plate count using the Foley–Dorsey method). The corresponding kinetic plots, however, indicate that the much reduced permeability of the second generation monoliths results in kinetic performances (time needed to achieve a given efficiency) which are only better than those of the first generation for plate counts up to N∼45,000. For more complex samples (N≥50,000), the first generation monoliths can intrinsically still provide faster analysis due to their high permeability. It is also demonstrated that – despite the improved efficiency of the second generation monoliths in the practical range of separations (N=10,000–50,000) – these columns can still not compete with state-of-the-art core–shell particle columns when all columns are evaluated at their own maximum operating pressure (200bar for the monolithic columns, 600bar for core–shell columns). It is suggested that monolithic columns will only become competitive with these high efficiency particle columns when further improvements to their production process are made and their pressure resistance is raised.

A new N-hydroxyethyliminodiacetic acid modified core–shell silica phase for chelation ion chromatography of alkaline earth, transition and rare earth elements

Bare core–shell silica (1.7μm) has been modified with iminodiacetic acid functional groups via standard silane chemistry, forming a new N-hydroxyethyliminodiacetic acid (HEIDA) functionalised core–shell stationary phase. The column was applied in high-performance chelation ion chromatography and evaluated for the retention of alkaline earth, transition and heavy metal cations. The influence of nitric acid eluent concentration, addition of complexing agent dipicolinic acid, eluent pH and column temperature on the column performance was investigated. The efficiencies obtained for transition and heavy metal cations (and resultant separations) were comparable or better than those previously obtained for alternative fully porous silica based chelation stationary phases, and a similarly modified monolithic silica column, ranging from ∼15 to 56μm HETP. Increasing the ionic strength of the eluent with the addition of KNO3 (0.75M) and increasing the column temperature (70°C) facilitated the isocratic separation of a mixture of 14 lanthanides and yttrium in under 12min, with HETP averaging 18μm (7μm for Ce(III))

Rapid tea catechins and caffeine determination by HPLC using microwave-assisted extraction and silica monolithic column

A rapid reversed-phase high performance liquid chromatographic method using a monolithic column for the determination of eight catechin monomers and caffeine was developed. Using a mobile phase of water:acetonitrile:methanol (83:6:11) at a flow rate of 1.4mLmin−1, the catechins and caffeine were isocratically separated in about 7min. The limits of detection and quantification were in the range of 0.11–0.29 and 0.33–0.87mgL−1, respectively. Satisfactory recoveries were obtained (94.2–105.2±1.8%) for all samples when spiked at three concentrations (5, 40 and 70mgL−1). In combination with microwave-assisted extraction (MAE), the method was applied to the determination of the catechins and caffeine in eleven tea samples (6 green, 3 black and 2 oolong teas). Relatively high levels of caffeine were found in black tea, but higher levels of the catechins, especially epigallocatechin gallate (EGCG) were found in green teas.