Liquid Chromatography Mode Research Articles

Mussel-inspired polydopamine-assisted hydroxyapatite as the stationary phase for capillary electrochromatography

A novel capillary with hydroxyapatite (HAP) as the stationary phase was prepared for open-tubular capillary electrochromatography (OT-CEC). To immobilize HAP, a mussel-inspired polydopamine method was utilized to modify the capillary firstly, generating a polydopamine layer; and then a layer of HAP would be formed on the polydopamine layer by a biomineralization process, to produce a HAP-modified capillary (HAP@capillary). Scanning electron microscopy (SEM), energy dispersive X-ray spectrometry (EDS) and X-ray photoelectron spectroscopy (XPS) provided evidence of nanostructured HAP grown on the surface of the capillary wall. The electroosmotic flow (EOF) characteristic of the HAP@capillary was investigated by varying the percentage of acetonitrile and pH value of the buffer with thiourea as a marker, and a pH-dependent EOF from anode to cathode was observed. The HAP@capillary exhibits high column efficiency for methylbenzene, up to 151,138 plates per meter. Different kinds of compounds including alkylbenzenes, phenols and amines have been successfully separated by the HAP@capillary in CEC mode. The HAP@capillary also possessed good separation ability in capillary liquid chromatography (CLC) mode because of the relatively large ratio of HAP in the capillary; however, the separation efficiency was not as good as that in CEC mode. The reproducibilities of the HAP@capillary were evaluated, and the relative standard deviations were found to be lower than 5%.

Synthesis and chromatographic evaluation of sulfobetaine-based capillary zwitterionic hydrophilic monolithic column using a binary porogenic agent of polyethylene glycol/methanol

Sulfobetaine-based capillary zwitterionic hydrophilic monolithic columns were synthesized with a novel binary porogenic agent of polyethylene glycol (PEG)/methanol. The polymer was prepared with (3-(metharyloylamino) propyl) dimethyl (3-sulfopropyl) ammonium hydroxide inner salt (SPP) as monomer, pentaerythritol triacrylate (PETA) as crosslinker, and azobisisobutyronitrile (AIBN) as initiator. In order to optimize the properties, the contents of the polymerization mixture were investigated. The optimum preparation conditions were as follows: the mass ratio of monomer and porogenator = 1:2.5; the mass ratio of SPP and PETA = 1:1 in the monomer; the mass ratio of PEG and methanol = 2:1 in the binary porogenic agent; the content of the initiator (AIBN) = 0.1% (m/m). With the binary porogenic agent of PEG/ methanol addition, good mechanical stability, homogeneous column bed, good permeability and narrow pore size distribution were obtained. In the capillary liquid chromatography mode, the hydrophilic monolith provided column efficiency up to 2.4 x 10(5) plates/m which was much higher than that fabricated by traditional method without PEG/methanol. The columns were used in capillary liquid chromatography and pressurized capillary electrochromatography for the separation of a mixture of phenols, nucleosides and so on.

Peak Distortion Effects in Analytical Ion Chromatography

The elution profile of chromatographic peaks provides fundamental understanding of the processes that occur in the mobile phase and the stationary phase. Major advances have been made in the column chemistry and suppressor technology in ion chromatography (IC) to handle a variety of sample matrices and ions. However, if the samples contain high concentrations of matrix ions, the overloaded peak elution profile is distorted. Consequently, the trace peaks shift their positions in the chromatogram in a manner that depends on the peak shape of the overloading analyte. In this work, the peak shapes in IC are examined from a fundamental perspective. Three commercial IC columns AS16, AS18, and AS23 were studied with borate, hydroxide and carbonate as suppressible eluents. Monovalent ions (chloride, bromide, and nitrate) are used as model analytes under analytical (0.1 mM) to overload conditions (10-500 mM). Both peak fronting and tailing are observed. On the basis of competitive Langmuir isotherms, if the eluent anion is more strongly retained than the analyte ion on an ion exchanger, the analyte peak is fronting. If the eluent is more weakly retained on the stationary phase, the analyte peak always tails under overload conditions regardless of the stationary phase capacity. If the charge of the analyte and eluent anions are different (e.g., Br(-) vs CO3(2-)), the analyte peak shapes depend on the eluent concentration in a more complex pattern. It was shown that there are interesting similarities with peak distortions due to strongly retained mobile phase components in other modes of liquid chromatography.

Study of hydration process on silica hydride surfaces by microcalorimetry and water adsorption

A series of hydrosilated stationary phases were compared with respect to their hydrophilic–hydrophobic properties. The stationary phases were also compared to the bare silica gel used for this synthesis. The investigations were done using microcalorimetric measurements of methanol and acetonitrile heats of immersion. Because these stationary phases are used in both the reversed-phase and aqueous normal phase modes of liquid chromatography, the excess isotherm of water from acetonitrile solution was measured.From the materials tested the highest polarity was exhibited by the silica hydride and the bare silica. The Diamond Hydride is less polar. The highest hydrophobicity is exhibited by the hydrosilated stationary phase which contains bonded octadecyl ligands.

Separation of benzoxazine enantiomers on β-cyclodextrin bonded chiral stationary phases

Chiral separation of benzoxazine enantiomers was studied on click chemistry based beta-cyclodextrin stationary phases under reversed-phase high performance liquid chromatographic mode. The effects of the type and percentage of the organic modifier, the concentration of triethylammonium acetate buffer (TEAA), and pH on enantioselective separation were examined and studied. It was demonstrated that acetonitrile was better for the chiral separation of benzoxazine enantiomers than methanol. It was observed that the retention time and the resolution of benzoxazine enantiomers decreased with the increase of the volume ratio of TEAA from 0.1% to 1.0%. The separation of benzoxazine enantiomers was of the maximum resolution at pH 4.1. With the optimized mobile phase of the mixture of acetonitrile and 0. 1% TEAA (pH 4.1), all the enantiomers were separated at the baseline. The chiral recognition mechanism is also discussed. The separation was probably based on the inclusion complex interaction and the hydrogen bonding between enantiomers and chiral stationary phases. This work provided the experience for the intensive study of click beta-cyclodextrin bonded stationary phases, and it also illustrated the potential of click chemistry in the preparation of beta-cyclodextrin based chiral stationary phase.

Preparation, characterization, and application of a new stationary phase containing different kinds of amine groups

A new stationary phase containing primary amine and tertiary amine groups was synthesized and its structure was confirmed by infrared spectra, elemental analysis, and zeta potential. This new material was packed into a stainless steel column and the effect of acetonitrile (ACN) content in mobile phase on retention was evaluated using probes of nucleosides, sulfanilamides, and quinolone compounds. This showed hydrophilic interaction, ion interaction, or a mixed-mode retention mechanism for different kinds of solutes, respectively. The effects of the pH and ion strength of the mobile phase were investigated to further understand the retention mechanism of the new stationary phase. The interaction forces caused by the new phase included adsorption, electrostatic function, and ion exchange. The new material could be used as a stationary phase in different high-performance liquid chromatography modes.

Preparation and chromatographic evaluation of a novel phosphate ester-bonded stationary phase with complexation and hydrophobic interactions retention mechanism

The present study described the preparation of a novel phosphate ester-bonded silica (PES) stationary phase based on “thiol-ene” click chemistry. The composition of the surface grafts of PES stationary phase was determined by elemental analysis and solid state 31P MAS NMR. Due to its hydrophilic phosphate-ester groups and short hydrophobic alkyl chains, the PES stationary phase exhibited dual retention mechanism via complexation and hydrophobic interactions with phenols. Benefiting from this special interaction mechanism, the newly synthesized PES stationary phase showed better selectivity in the separation of phenols compared to commercial octadecylsilyl-bonded silica (C18) columns. Furthermore, the separations of 10 nucleosides and nucleobases on the PES stationary phase were achieved in both reversed-phase liquid chromatography (RPLC) mode and hydrophilic interaction liquid chromatography (HILIC) mode.

SDS-Based Biomembrane Mimetic Chromatography for Prediction of Human Drug Transport as an in Vitro Technique

The main oral drug absorption barriers are fluid cell membranes, and generally drugs are absorbed by a passive diffusion mechanism. On the other hand, the blood–brain barrier (BBB) is considered to be the main barrier to drug transport into the central nervous system (CNS). The BBB restricts the passive diffusion of many drugs from blood to brain. Biopartitioning micellar chromatography (BMC), a mode of micellar liquid chromatography that uses micellar mobile phases in adequate experimental conditions, can be useful as an in vitro system in mimicking the drug partitioning process into biological systems. In this study, relationships between the BMC retention data of a heterogeneous set of 12 drugs and their pharmacokinetics parameters (human oral drug absorption and BBB penetration ability) are studied and the predictive ability of the models is evaluated. Modeling of log kBMC of these compounds was established by multiple linear regression in two different concentrations (0.07 and 0.09 M) of sodium dodecyl sulfate (SDS). The results showed a fair correlation between human oral drug absorption and BMC retention data in 0.09 M SDS (R2 = 0.864) and a good correlation between the blood–brain distribution coefficient and BMC retention data in 0.07 M of SDS (R2 = 0.887). Application of the developed models to a prediction set demonstrated that the model is also reliable with good predictive accuracy. The external and internal validation results showed that the predicted values are in good agreement with the experimental value.

Bidentate urea-based chiral selectors for enantioselective high performance liquid chromatography: Synthesis and evaluation of “Crab-like” stationary phases

A rational approach for the design and preparation of two new “Crab-like” totally synthetic, brush-type chiral stationary phases is presented. Enantiopure diamines, namely 1,2-diaminocyclohexane and 1,2-diphenyl-1,2-ethylene-diamine were treated with 3-(triethoxysilyl)propyl isocyanate, to yield reactive ureido selectors that were eventually attached to unmodified silica particles through a stable, bidentate tether, through a facile two-step one-pot procedure. A full chemical characterization of the new materials has been obtained through solid-state NMR (both 29Si and 13C CPMAS) spectroscopy. Columns packed with the two Crab-like chiral stationary phases allow for different mechanisms of separation: normal phase liquid chromatography, reversed phase liquid chromatography and polar organic mode and show a high stability at basic pH values. In particular, the Crab-like column containing the 1,2-diphenyl-1,2-ethylene-diamine selector proved a promising candidate for the resolution of a wide range of racemates (including benzodiazepines, N-derivatized amino acids, and free carboxylic acids) both in normal phase and polar organic mode. An Hmin of 9.57 at a μsf of 0.80mm/s (corresponding to 0.8mL/min) was obtained through van Deemter analysis, based on toluene, for the Crab-like column with the 1,2-diphenyl-1,2-ethylene-diamine selector (250mm×4.6mm I.D.), with a calculated reduced height equivalent to a theoretical plate (h) of only 1.91. Finally, comparative studies were performed with a polymeric commercially available P-CAP-DP column in order to evaluate enantioselectivity and resolution of the Crab-like columns.

Comparison between the intra-particle diffusivity in the hydrophilic interaction chromatography and reversed phase liquid chromatography modes. Impact on the column efficiency

The effective diffusion coefficients of five low molecular weigh compounds (naphthalene, uracil, uridine, adenosine, and cytosine) were measured at room temperature in a 4.6mm×100mm column packed with 3.5μm XBridge HILIC particles. The mobile phase was an acetonitrile–water mixture (92.5/7.5, v/v) containing 10mM ammonium acetate and 0.02% acetic acid. Using a physically reliable model of effective diffusion in binary composite media (Torquato's model), accurate estimates of the intra-particle diffusivities in the HILIC particles were obtained as a function of the retention of these analytes. The HILIC diffusion coefficients were compared to those previously obtained for endcapped RPLC-C18 particles (5.0μm Gemini-C18). The experimental results confirm that adsorption sites are not localized in RPLC whereas they are so in the HILIC mode. In contrast to RPLC columns, HILIC columns provide longitudinal diffusion B/u terms that increase very little with increasing retention factors. This confirms the absence of surface diffusion in HILIC. The impact of intra-particle diffusivity on the column efficiency was projected in HILIC and RPLC on the basis of the measured intra-particle diffusivities and on the well established theory of band broadening in particulate columns. Accordingly, RPLC columns generate short-range eddy dispersion and solid–liquid mass transfer resistance Cu terms that increase less than do HILIC column with increasing retention factors. The HETP contribution caused by the trans-column structure heterogeneity is smaller in the HILIC than in the RPLC modes because the transverse excursion length is smaller in HILIC. Even though the overall column efficiencies are comparable in HILIC and RPLC, this study shows that the individual mass transfer phenomena are inherently different in the HILIC and the RPLC retention modes.

Application of Urine Metabolomics to Probe the Effects of Long‐Term Bed Rest as a Model for Spaceflight

This project aims to determine if urine metabolomics can be a meaningful approach to assess spaceflight health effects. Muscle loss in spaceflight might be caused by decreased muscle synthesis or increased muscle breakdown, and identification of an appropriate biomarker might suggest appropriate countermeasures. In this study, urine samples from 8 subjects in a bed rest study (paired before and during bed rest) were analyzed using hydrophilic interaction liquid chromatography (HILIC) and positive ionization mode time‐of‐flight mass spectrometry. Data analysis indicated measurable differences in urinary compounds of the paired groups. Of 1873 unique compounds found, 20 were identified as having high significance (consistent increase or decrease during the sample period) and were thus considered possible biomarkers. Comparison of the bioinformatics programs used here, Agilent Mass Profiler Professional and Purdue's in‐house Omics Discovery Pipeline, could be valuable for future metabolomic studies. Identification of high‐significance compounds is continuing. Support: Grant 10465 – Indiana Space Grant Consortium & Indiana University‐Purdue University Fort Wayne Office of Research & External Support (KLE).

Application of hydrophilic interaction chromatography in phytochemical analysis

Summary Hydrophilic interaction chromatography (HILIC) is a liquid chromatography technique when a polar stationary phases — silica or polar bonded phases and aqueous mobile phase containing amount of a less polar solvent (often acetonitrile) is applied. HILIC is important for the separation of highly polar substances including biologically active compounds, such as drugs, amino acids, peptides, proteins, nucleosides, neurotransmitters, etc. HILIC is also the appropriate method for analysis of plant extract polar components such as carbohydrates, amino acids, peptides, phenolic acids, flavonoids and some alkaloids. Plant extracts are usually multicomponent mixtures of compounds of wide polarity range, which often cannot be analyzed by use of a single separation and detection method because of the high chemical diversity. Good results are obtained by use of coupled method, e.g., reversed-phase liquid chromatography (RP-LC) and HILIC mode separation. The elution order in HILIC is usually opposite to that in RP separations. This orthogonality determines one of the advantages of HILIC — the possibility of creating multidimensional separations. An important feature of HILIC is the improved sensitivity with electrospray mass spectrometry. This is significant for the analysis of components existing in small concentration in multicomponent mixtures. The high acetonitrile content also gives additional advantage of HILIC — faster separations due to the lower viscosity of HILIC eluents compared to standard RP ones. The presented review deals with the optimization of HILIC separations and application of the method for analysis of plant extracts components.

The use of biopartitioning micellar chromatography and immobilized artificial membrane column for in silico and in vitro determination of blood–brain barrier penetration of phenols

Biopartitioning Micellar Chromatography (BMC) is a mode of micellar liquid chromatography that uses C18 stationary phases and micellar mobile phases of Brij35 under adequate experimental conditions and can be useful to mimic human drug absorption, blood–brain barrier distribution or partitioning processes in biological systems. BMC system can be useful in constructing good predictive models because the characteristics of the BMC system are similar to biological barriers and extracellular fluids. Immobilized Artificial Membrane (IAM) chromatography uses stationary phase which consists of a monolayer of phosphatidylcholine covalently immobilized on an inert silica support. IAM columns are thought to mimic very closely a membrane bilayer and are used in a HPLC system with a physiological buffer as eluent. In this paper the usefulness of BMC and IAM system for in silico and in vitro determination of blood–brain barrier (BBB) penetration of phenols has been demonstrated. The most important pharmacokinetic parameters of brain have been obtained for the determination of BBB penetration, i.e. BBB permeability – surface area product (PS), usually given as a logPS, brain/plasma equilibration rate (log(PS×fu,brain)) and fraction unbound in plasma (Fu). Moreover, the relationships between retention of eighteen phenols and different parameters of molecular size, lipophilicity and BBB penetration were studied. Extrapolated to pure water values of the logarithms of retention factors (logkw) have been compared with the corresponding octanol–water partition coefficient (logPo–w) values of the solutes. In addition, different physicochemical parameters from Foley's equation for BMC system have been collated with the chromatographic data. The Linear Solvation Energy Relationship (LSER) using Abraham model for the describing of phenols penetration across BBB has been used. Four equations were developed as a multiple linear regression using retention data from IAM and BMC system (QRAR models) and molecular volume parameter (Vm) and Abraham descriptors to correlate the logBB values. Moreover, in order to establish the relationships between different variables, the principal components analysis (PCA) has been done. The results of PCA were obtained using chromatographic data from IAM and BMC systems as well as from the structures of tested phenols. The four parameters: logkwIAM(exp), logkwBMC(exp), analyte-micelle association constant (Kma) and logPo–w have been checked.

Purification of saponins from leaves of Panax notoginseng using preparative two-dimensional reversed-phase liquid chromatography/hydrophilic interaction chromatography

Saponins are widely distributed in the plant kingdom and have been shown to be active components of many medicinal herbs. In this study, a two-dimensional purification method based on reversed-phase liquid chromatography coupled with hydrophilic interaction liquid chromatography was successfully applied to purify saponins from leaves of Panax notoginseng. Nine saponin reference standards were used to test the separation modes and columns. The standards could not be resolved using C18 columns owing to their limited polar selectivity. However, they were completely separated on a XAmide column in hydrophilic interaction liquid chromatography mode, including two pairs of standards that were coeluted on a C18 column. The elution order of the standards on the two columns was sufficiently different, with a correlation coefficient between retention times on the C18 and XAmide columns of 0.0126, indicating good column orthogonality. Therefore, the first-dimension preparation was performed on a C18 column, followed by a XAmide column that was used to separate the fractions in the second dimension. Fifty-four fractions were prepared in the first dimension, with 25 fractions rich in saponins. Eight saponins, including two pairs of isomeric saponins and one new saponin, were isolated and identified from three representative fractions. This procedure was shown to be an effective approach for the preparative isolation and purification of saponins from leaves of P. notoginseng. Moreover, this method could possibly be employed in the purification of low-content and novel active saponins from natural products.

Influence of pH value of mobile phase on phosphopeptide enrichment selectivity under hydrophilic interaction liquid chromatography mode by using Click OEG-CD matrix

The digest of the standard protein alpha-casein was used as the objective to investigate the influence of pH value of mobile phase on phosphopeptide enrichment selectivity by using Click OEG-CD matrix. Disodium phenyl phosphate was primarily selected as the model sample. The results indicated that phosphate group was difficultly to be ionized when the pH value of mobile phase was below its pKa value. The retention of disodium phenyl phosphate on the Click OEG-CD matrix was not based on ion exchange interaction, leading to the weak retention. The influence of pH value (2, 4 and 6) of mobile phase under hydrophilic interaction liquid chromatography (HILIC) mode on phosphopeptide enrichment selectivity was investigated taking the digest of alpha-casein as a sample. Phosphopeptides could not be enriched on the matrix with the pH value of 2; phosphopeptides could be enriched on the matrix and the elution window was narrow with the pH value of 4; phosphopeptides could be enriched on the matrix while the elution window was broad with the pH value of 6. According to the investigation, Click OEG-CD matrix could be better applied in phosphopeptide enrichment.

Nano-particle modified stationary phases for high-performance liquid chromatography

This review covers the latest developments and applications of nano-materials in stationary phase development for various modes of high-performance liquid chromatography. Specific attention is placed upon the development of new composite phases, including the synthetic and immobilisation strategies used, to produce either encapsulated nano-particles, or surface attached nano-particles, layers, coatings and other structures. The resultant chromatographic applications, where applicable, are discussed with comment upon enhanced selectivity and/or efficiency of the nano-particle modified phases, where such effects have been identified. In the main this review covers developments over the past five years and is structured according to the nature of the nano-particles themselves, including carbonaceous, metallic, inorganic, and organopolymer based materials.

Metabolic Profiling and Quantification of Sphingolipids by Liquid Chromatography-Tandem Mass Spectrometry

A precise, robust, and specific liquid chromatography-electrospray tandem mass spectrometry (LC-ESI-MS/MS) method was developed for profiling and quantifying glucosyl sphingosine (GS), glucosylceramide (GC), ceramide (Cer), lactosylceramide (LacCer) and sulfatide lipid species in a variety of mouse tissues. The linear response ranges of these species were 0.05-25 ng. The major GC species identified in visceral tissues of mice were GCs with N-acyl chains of C24-1, C24, C22, C16 lengths, but the qualitative and quantitative profiles differed among tissues. GC levels in spleen were approximately 3-5 times higher than in liver and lung. Brain differed from visceral tissues in that galactosylceramides (GalCer) were the predominant monohexosylceramide species identified. A silica column used in hydrophobic interaction liquid chromatography (HILIC) mode was capable of differentiating GC and GalCer. The analysis of mouse brain samples revealed that GC accounted for only 0.3% of the total monohexosylceramides. Cer and LacCer were also profiled and quantified in mouse brain, lung, liver and spleen. Application of these methods greatly facilitated a range of targeted sphingolipidomic investigations and will permit a better understanding of the function and mechanism of these diverse molecular species in various disease animal models, including Gaucher disease.

Synthesis of porous spherical silicon oxynitride material and evaluation of its properties in reversed-phase chromatographic separation

Silica has been widely used as HPLC column packing material. However, the fact that base can attack the silanol and dissolve the silica embarrasses the utilization of silica stationary phase in high pH mobile phases (pH >8). In our previous research, the use of porous spherical silicon oxynitride (sph-SiON) material from high temperature nitridation of silica microspheres as stationary phase for HPLC has been explored, and the sph-SiON is stable to alkaline mobile phases and demonstrates excellent separation of a variety of polar compounds in hydrophilic interaction liquid chromatography (HILIC) mode. Herein, the degree of nitridation was studied as a function of temperature of nitridation at 750-1 050 degrees C, yielding the silicon oxynitride with 0.40%-12.0% (mass fraction) nitrogen from elemental analysis. At the temperature of 1 050 degrees C, the nitrogen content increased from 12.0% to 24.5% with the nitridation time increasing from 20 h to 120 h. The sph-SiON is stable when disposed in different pH aqueous solutions for one week. The sph-SiON material can be modified to give hydrophobic surface through the reaction of surface Si-NHx with dimethyloctadecylchlorosilane. Elemental analysis and 13C cross-polarization magic-angle spinning (CP/MAS) NMR spectrum of C18-sph-SiON prove the integration of C18 alkyl groups attached onto the sph-SiON surface. The chromatographic evaluation of C18-sph-SiON in reversed-phase separation mode was performed with alkylbenzenes as hydrophobic probes. Three alkylbenzene compounds can be separated and retained well on C18-sph-SiON even in the mobile phase of methanol/H2O (70/30, v/v) with 78 507 plates/m, and an excellent tailing factor (0.95) can be obtained for ethylbenzene. In comparison with C18-SiO2, C18-sph-SiON shows distinct differences with respect to different classes of analytes, i. e. neutral analyte naphthalene, acidic analyte ibuprofen, and basic analyte amitriptyline.

Chromatographic Behavior of Aromatic Diamines in Hydro-Organic, Micellar and Submicellar Reversed Phase Liquid Chromatographic Modes

The chromatographic behavior (retention, elution strength, efficiency, peak asymmetry and selectivity) of some aromatic diamines in the presence of methanol with or without anionic surfactant SDS in the four different reversed phased liquid chromatographic (RPLC) modes, i.e., hydro-organic, micellar (MLC), low submicellar (LSC) and high submicellar (HSC), was investigated. In the three surfactant-mediated modes, the surfactant monomers coat the stationary phase even up to 70 % methanol; this results in the suppression of peak tailing (by masking the silanol groups on the stationary phase). In MLC and HSC, the solute retention decreases by increasing the surfactant concentration, while this situation was reversed in LSC. In the region between MLC and HSC modes (25–50 % methanol), retention of late eluting solutes was increased by increasing methanol content which is seemingly due to disaggregation of SDS micelles. Changes in selectivity were observed after changing the concentrations of SDS and methanol, in a greater extent when concentration of SDS was changed. Among the four studied RPLC modes, HSC showed the best efficiency with nearly symmetrical peaks. Prediction of retention of solutes in HSC based on a mechanistic retention model combined with Pareto-optimality method allowed the full resolution of target diamines in practical analysis times.

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.