Silica-based Column Research Articles

Development of a novel comprehensive analytical method for volatile compounds using supercritical fluid chromatography/mass spectrometry with a highly cross-linked styrene divinylbenzene polymer-based column.

Analytical techniques to determine volatile compounds such as flavor, aroma, and fragrances are in high demand due to their wide range of applications in industry, the chemical properties of them are very diverse. Supercritical fluid chromatography (SFC) is capable of high speed, high peak capacity separation and has a high separation coverage. It is also an advantageous for preparative purifications due to its unique mobile phase conditions. However, there is no column commercially available for SFC that is suitable to comprehensively separate volatile compounds. SFC is limited to the use of silica-based columns due to weak retentions and polymer-based column issues such as pressure, swelling and shrinkage tolerances. This study demonstrated comprehensive analytical method for volatile in SFC using a highly cross-linked styrene divinylbenzene (SDVB) polymer-based column, newly developed for SFC. In this study, 23 typical volatile compounds with a wide variety of chemical properties were selected as model compounds. The newly developed SDVB column showed, compared to conventional silica-based columns (k > 0.3), an excellent overall and substantial improved retentions (k > 1.6) under SFC mobile phase conditions. It was also able to retain esters (hydroxy acetate, pentyl butylate, methyl salicylate) and non-polar terpenes (limonene, pinene) that did not show sufficient retention in any other commercially available silica-based columns. Aldehydes reacting on NH2 column due to Schiff base formation were also successfully eluted. It was confirmed that SDVB column provided comprehensive separation and wide coverage for volatile compounds.

Hydrophilic Liquid Chromatography versus Reversed-Phase Liquid Chromatography in the Absence and the Presence of 1-Hexyl-3-methylimidazolium Chloride for the Analysis of Basic Compounds

In reversed-phase liquid chromatography (RPLC), positively charged basic compounds yield broad and asymmetric peaks, as a result of ionic interactions with free silanols that remain on conventional silica-based columns. Diverse solutions have been proposed to mask the silanophilic activity, which is translated to an improved peak shape. In this work, the chromatographic performance of hydrophilic interaction liquid chromatography (HILIC) was evaluated as an alternative to the addition of an ionic liquid (IL) to the aqueous-organic mobile phase used with RPLC columns, for the analysis of eight β-adrenoceptor antagonists. ILs change the behavior of RPLC stationary phases owing to adsorption on their surface. Meanwhile, in HILIC, a layer of adsorbed water is formed on the stationary phase surface. The association of cationic basic compounds with the adsorbed additive ions, hydrophilic partitioning on the HILIC columns, and other interactions, give rise to complex retention mechanisms. The chromatographic behavior was examined in terms of retention, elution strength, selectivity, peak shape and resolution, using acetonitrile-water mobile phases buffered at pH 3. Both chromatographic modes, RPLC with added IL and HILIC, proved to be a viable solution to the problem of poor peak shape for basic compounds.

Allelic discrimination between circulating tumor DNA fragments enabled by a multiplex-qPCR assay containing DNA-enriched magnetic ionic liquids

Multiplex amplification of DNA can be highly valuable in circulating tumor DNA (ctDNA) analysis due to the sheer number of potential mutations. However, commercial ctDNA extraction methods struggle to preconcentrate low concentrations of DNA and require multiple sample handling steps. Recently, magnetic ionic liquids (MILs) have been used to extract DNA and were integrated with a quantitative polymerase chain reaction (qPCR). However, in previous studies, DNA could not be preconcentrated from plasma and only one fragment could be amplified per reaction. In this study, MILs were utilized as DNA extraction solvents and directly integrated into a multiplex-qPCR buffer to simultaneously amplify wild-type KRAS, G12S KRAS, and wild-type BRAF, three clinically-relevant genes whose mutation status can affect the success of anti-EGFR therapy. DNA was desorbed from the MIL solvent during a multiplex-PCR without having a significant effect on the amplification efficiency, and allelic discrimination of single-nucleotide polymorphisms could still be achieved. Enrichment factors over 35 for all three sequences were achieved from Tris buffer using the [N8,8,8,Bz+][Ni(hfacac)3-]) and [P6,6,6,14+][Ni(Phtfacac)3-] MILs. DNA could still be preconcentrated from 2-fold diluted human plasma using the [N8,8,8,Bz+][Ni(hfacac)3-] MIL. Extractions from undiluted plasma were reproducible with the [P6,6,6,14+][Ni(Phtfacac)3-] MIL although DNA was not preconcentrated with enrichment factors around 0.6 for all three fragments. Compared to commercial DNA extraction methods (i.e., silica-based spin columns and magnetic beads), the MIL-based extraction achieved higher enrichment factors in Tris buffer and plasma. The ability of the MIL-based dispersive liquid-liquid microextraction (DLLME) direct-multiplex-qPCR method to simultaneously achieve high enrichment factors of multiple DNA fragments from human plasma is highly promising in the field of ctDNA detection.

Emulsion PCR made easy.

Emulsion PCR (ePCR) is an important technique that permits amplification of DNA molecules in physically separated picoliter-volume water-in-oil droplets, and thus avoids formation of unproductive chimeras and other artifacts between similar DNA sequences. However, the recovery of ePCR products involves repeated extraction with hazardous organic solvents followed by purification using silica-based columns, making the overall process cumbersome.In this benchmark, we have described a quick ePCR extraction protocol for the purification of ePCR products, which directly employs silica-based DNA purification columns; products purified using this method have been found to be compatible with gene cloning and next-generation sequencing applications. The method described here makes ePCR easy, safe and within the reach of every laboratory.

Evaluating the advantages of higher heat conductivity in a recently developed type of core-shell diamond stationary phase particle in UHPLC

In recent studies, the nature and magnitude of the temperature gradients developed in ultra-high pressure liquid chromatography (UHPLC), were found to be dependent on the heat conductivity properties of the column matrices, but also, on the principle used for controlling the temperature over the column. Here, we investigated the potential of using highly heat conductive diamond-based stationary phases (85 times higher than silica), for reducing the temperature gradients. The stationary phases investigated were a (i) Diamond Analytics FLARE column, based on particles comprised of a graphite core surrounded by a very thin diamond shell, and two silica hybrid columns: (ii) a core-shell silica Kromasil Eternity Shell column and (iii) a fully porous silica Kromasil Eternity XT column. Models were developed based on two-dimensional heat transfer theory and mass transfer theory, which were used to model the temperature profiles and the migration of an analyte band accounting for column efficiencies at different flow rates. For the silica-based columns, using water-controlled temperature mode, the temperature gradients along the column axes are suppressed whereas temperature gradients in the radial direction prevails resulting in decreased column efficiencies. Using these columns with air-controlled temperature mode, the radial temperature gradients are reduced whereas temperature gradients along the column prevails resulting in decreased retention times. With the Diamond FLARE column, there was no loss in column efficiency using the water-controlled temperature mode and the van Deemter curves are almost identical using both temperature control modes. Thus, for the Diamond FLARE column, in contrast to the silica-based columns, there are almost no losses of column efficiencies due to reduced radial temperature gradients independent on how the column temperature was controlled.

Separation of New Coumarin Glycosides from Toddalia asiatica Using Offline Two-Dimensional High-Performance Liquid Chromatography.

Coumarins and flavonoids are the major constituents of Toddalia asiatica. The separation and purification of ingredients from T. asiatica is an important procedure to acquire high-purity compounds for subsequent pharmacological investigation to discover leading compounds. In the present work, an offline two-dimensional high-performance liquid chromatography (HPLC) method was successfully established for the separation of high-purity glycosides from T. asiatica. Based on the separation results obtained with two different chromatographic stationary phases, a phenyl-bonded silica-based reversed-phase column was employed as the first HPLC preparation, and three fractions were obtained from the sample. Then, the fractions were isolated and purified on an octadecyl-bonded silica-based reversed-phase column to obtain high-purity compounds in the second HPLC separation. As a result, three coumarin glycosides, including two undescribed and one known, along with one known flavonoid glycoside with more than 98% purity were isolated from the sample. The structures of the isolated compounds were elucidated on the basis of extensive spectroscopic evidence derived from optical rotation, mass spectrometry, and nuclear magnetic resonance experiments. Two-dimensional HPLC with different stationary phases has the potential to be an efficient method for the separation of high-purity compounds from T. asiatica.

Evaluation of mitochondrial DNA copy number estimation techniques.

Mitochondrial DNA copy number (mtDNA-CN), a measure of the number of mitochondrial genomes per cell, is a minimally invasive proxy measure for mitochondrial function and has been associated with several aging-related diseases. Although quantitative real-time PCR (qPCR) is the current gold standard method for measuring mtDNA-CN, mtDNA-CN can also be measured from genotyping microarray probe intensities and DNA sequencing read counts. To conduct a comprehensive examination on the performance of these methods, we use known mtDNA-CN correlates (age, sex, white blood cell count, Duffy locus genotype, incident cardiovascular disease) to evaluate mtDNA-CN calculated from qPCR, two microarray platforms, as well as whole genome (WGS) and whole exome sequence (WES) data across 1,085 participants from the Atherosclerosis Risk in Communities (ARIC) study and 3,489 participants from the Multi-Ethnic Study of Atherosclerosis (MESA). We observe mtDNA-CN derived from WGS data is significantly more associated with known correlates compared to all other methods (p < 0.001). Additionally, mtDNA-CN measured from WGS is on average more significantly associated with traits by 5.6 orders of magnitude and has effect size estimates 5.8 times more extreme than the current gold standard of qPCR. We further investigated the role of DNA extraction method on mtDNA-CN estimate reproducibility and found mtDNA-CN estimated from cell lysate is significantly less variable than traditional phenol-chloroform-isoamyl alcohol (p = 5.44x10-4) and silica-based column selection (p = 2.82x10-7). In conclusion, we recommend the field moves towards more accurate methods for mtDNA-CN, as well as re-analyze trait associations as more WGS data becomes available from larger initiatives such as TOPMed.

Lipidomic characterization of exosomes isolated from human plasma using various mass spectrometry techniques

Ultrahigh-performance supercritical fluid chromatography - mass spectrometry (UHPSFC/MS), ultrahigh-performance liquid chromatography - mass spectrometry (UHPLC/MS), and matrix-assisted laser desorption/ionization (MALDI) - MS techniques were used for the lipidomic characterization of exosomes isolated from human plasma. The high-throughput methods UHPSFC/MS and UHPLC/MS using a silica-based column containing sub-2 μm particles enabled the lipid class separation and the quantitation based on exogenous class internal standards in <7 minute run time. MALDI provided the complementary information on anionic lipid classes, such as sulfatides. The nontargeted analysis of 12 healthy volunteers was performed, and absolute molar concentration of 244 lipids in exosomes and 191 lipids in plasma belonging to 10 lipid classes were quantified. The statistical evaluation of data included principal component analysis, orthogonal partial least square discriminant analysis, S-plots, p-values, T-values, fold changes, false discovery rate, box plots, and correlation plots, which resulted in the information on lipid changes in exosomes in comparison to plasma. The major changes were detected in the composition of triacylglycerols, diacylglycerols, phosphatidylcholines, and lysophosphatidylcholines, whereby sphingomyelins, phosphatidylinositols, and sulfatides showed rather similar profiles in both biological matrices.

Simplified, rapid DNA extraction protocol for STR typing from bones

Bones are one of the body parts most tolerant towards various environmental conditions, especially as unidentified remains found in several circumstances: mass disaster, terrorism, war, and plague. Typically, the most complex and time-consuming part of the STR typing process is DNA extraction, with many different methods developed, such as organic extraction, total demineralization (TD), and silica-based column extraction. These protocols may take more than a day to complete. In such events where time is of the essence for human identification of the remains, a simple and fast but effective method is required. This study aimed to develop a simplified and rapid DNA extraction protocol from bone samples. Two bone types (fresh femur and tibia) were incubated in a buffer of SLS, EDTA, proteinase K, and DTT. We also evaluated extraction from different amounts of bone powder. Comparison was done for the developed method against standard total demineralisation protocol. Quantity of DNA extracted was slightly lower for the developed protocol compared to TD, however this did not affect STR typing and profile interpretability. DNA extraction was also possible from merely 0.1 g of bone. The developed method used significantly shorter incubation time and processing steps. Our final protocol only required two hours of incubation and 0.1 g of bone powder. STR typing results gave full profiles. The rapid protocol could be used when there is an urgent need of STR typing for human identification or when the sample amount is limited with a significant reduction in cost and time for investigation.

Applicability of a Monolithic Column for Separation of Isoquinoline Alkalodis from Chelidonium majus Extract.

Isoquinoline alkaloids are the main group of secondary metabolites present in Chelidonium majus extracts, and they are still the object of interest of many researchers. Therefore, the development of methods for the investigation and separation of the alkaloids is still an important task. In this work, the application potential of a silica-based monolithic column for the separation of alkaloids was assessed. The influence of the organic modifier, temperature, salt concentration, and pH of the eluent on basic chromatographic parameters such as retention, resolution between neighboring peaks, chromatographic plate numbers, and peak asymmetry were investigated. Based on the obtained results, a gradient elution program was developed and used to separate and quantitatively determine the main alkaloids in a Chelidonium majus root extract.

Separation and determination of amygdalin and unnatural neoamygdalin in natural food supplements by HPLC-DAD

ABSTRACTThis work is focused on separation and determination of amygdalin and its unnatural form neoamygdalin in natural food supplements. Reversed-phase high-performance liquid chromatography with a high-stability silica-based column with C18 functional group has been used for solving this problem. The effect of the mobile phase composition as well as the column temperature on the separation of the amygdalin epimers has been investigated. Isocratic elution using a mobile phase composed of 0.05% aqueous formic acid and acetonitrile achieved the required separation within 17 min. Under optimum chromatographic conditions, the developed method was validated and was applied for the determination of amygdalin epimers in natural food supplements containing apricot or peach kernels. A simple extraction method using methanol as an extractant supported by an ultrasonic bath was used with recovery in the range of 94.8% to 104.3%. The limit of detection and limit of quantification values for R-amygdalin were 0.13 mg/L and 0.40 mg/L, respectively. The developed method proved to be precise with the intra-day and inter-day relative standard deviation values less than 2.23%.

Hydrophilic Interaction Liquid Chromatography Coupled to Mass Spectrometry and Multivariate Analysis of the De Novo Pyrimidine Pathway Metabolites

In this study, we describe the optimization of a Hydrophilic Interaction Liquid Chromatography coupled to mass spectrometry (HILIC-MS) method for the evaluation of 14 metabolites related to the de novo synthesis of pyrimidines (dnSP) while using multivariate analysis, which is the metabolic pathway for pyrimidine nucleotide production. A multivariate design was used to set the conditions of the column temperature, flow of the mobile phase, additive concentration, gradient rate, and pH of the mobile phase in order to attain higher peak resolution and ionization efficiency in shorter analysis times. The optimization process was carried out while using factorial fractional designs, Box–Behnken design and central composite design while using two zwitterionic columns, ZIC-p-HILIC and ZIC-HILIC, polymeric, and silica-based columns, respectively. The factors were evaluated while using resolution (R), retention factor (k), efficiency of the column (N), and peak height (h) as the response variables. The best optimized conditions were found with the ZIC-p-HILIC column: elution gradient rate 2 min, pH 7.0, temperature 45 °C, mobile phase flow of 0.35 mL min−1, and additive (ammonium acetate) concentration of 6 mM. The total analysis time was 28 min. The ZIC-p-HILIC LC-MS method yielded satisfactory results for linearity of calibration curves, limit of detection (LOD), and limit of quantification (LOQ). The method has been shown to be appropriate for the analysis of dnSP on samples of tomato plants that were infected with Phytophthora infestans.

Monolithic Column Ion-Pair Chromatography with Indirect Ultraviolet Detection for the Determination of Three Acid Radical Anions Containing Fluorine in Ionic Liquids

A rapid ion-pair chromatographic method was developed on a monolithic column for the determination of trifluoroacetate, trifluoromethanesulfonate, and tetrafluoroborate. The three anions were successfully detected using indirect ultraviolet detection. These analyte anions were separated using a tetrabutylammonium hydroxide - p-toluenesulfonic acid – acetonitrile mobile phase on a silica-based monolithic column with an octadecyl functional group. The effects of detection wavelength, mobile phase, column temperature and flow rate on the retention of the anions were investigated. The chromatographic conditions were optimized and the retention properties were evaluated. Under the selected optimal conditions, the successful separation of the anions was achieved within 3.5 min. The detection limits were less than 1.0 mg/L for trifluoroacetate, trifluoromethanesulfonate, and tetrafluoroborate. The relative standard deviations for five repeated measurement of retention time and peak area were no more than 0.2%. The linear ranges extended across two orders of magnitude. The three anions in synthetic ionic liquid samples were successfully separated and detected by this method. The results show that the recoveries of this method were between 94.0% and 99.0%. The method was rapid, simple, and easy to use.

Enantioseparation of the racemates of a number of pesticides on a silica-based column with immobilized amylose tris(3-chloro-5-methylphenylcarbamate)

We report here the results from studies on the high-performance liquid chromatographic HPLC enantioresolution of thirty different types of pesticides on the chiral silica-based column with immobilized amylose tris(3-chloro-5-methylphenylcarbamate (ACMPC) (CHIRALPAK IG-3) under polar-organic and reversed-phase conditions measured at 20 °C. The main objective was to explore diverse polar and aqueous-based eluents for use with a single column to obtain an enantioseparation of polar pesticides usually found in environmental matrices. These pesticides included phenoxy- and aryloxyphenoxypropionic acids, imidazolinones, and diphenyl-ether herbicides; acylanilides, triazoles, and pyrimidine fungicides; chlordane and pyrethroid insecticides; organophosphorus compounds; and some metabolites and precursors. We obtained improved chromatographic enantioresolutions for seventeen of these compounds in either the polar-organic or reversed-phase modes, including six pesticides whose enantioseparation is reported for first time through the use of these chromatographic modes.

Reversed-phase liquid chromatography system constant database over an extended mobile phase composition range for 25 siloxane-bonded silica-based columns

The solvation parameter model is used to characterize the retention properties of 25 siloxane-bonded type-B silica columns under typical reversed-phase liquid chromatography conditions with binary aqueous mobile phases containing 10–70% (v/v) methanol and acetonitrile. To further explore the affect of solvent type on column selectivity complementary information is presented for aqueous phases containing 10–70% (v/v) tetrahydrofuran for two columns. Columns were selected to include examples of all common column packing morphologies for small molecule separations (totally porous particles, superficially porous particles, organic–inorganic hybrid particles, and a monolith) and common topologies octadecylsiloxane-bonded (including phases with a polar-embedded group, mixed-mode phases with a short-chain siloxane-bonded polar functional group, sterically crowded, positive shield, and fluorine-containing phases); octylsiloxane-bonded; and various phenyl-containing stationary phases with different linker arms, pentafluorophenylalkyl and biphenyl groups. The column properties are reported as a system constant database and as system maps for the full range of mobile phase compositions. Selectivity differences are evaluated using principal component factor analysis to classify the columns into selectivity groups and correlation plots of the system constants for compared columns at all mobile phase compositions. The later is shown to be a suitable technique to identify (near) selectivity equivalent columns and varied columns suitable for method development. Contributions to retention from steric resistance and cation-exchange interactions not parameterized in the solvation parameter model are identified for columns and conditions where they may be important. It is shown that the interaction parameters employed in the hydrophobic-subtraction model have little overlap with those of the solvation parameter model and a quantitative comparison of column properties delineated by both models is not possible.

Applications of monolithic columns in gas chromatography and supercritical fluid chromatography.

Porous monoliths are well-known stationary phases in high-performance liquid chromatography and capillary electrochromatography. Contrastingly, their use in other types of separation methods such as gas or supercritical fluid chromatography is limited and scarce. In particular, very few studies address the use of monolithic columns in supercritical fluid chromatography. These are limited to silica-based monoliths and will be covered in this review together with an underlying reason for this trend. The application of monoliths in gas chromatography has received much more attention and is well documented in two reviews by Svec and Kurganov published in 2008 and 2013, respectively. The most recent studies, covered in this review, build on the previous findings and on further understanding of the influence of preparation conditions on porous properties and chromatographic performance of poly(styrene-co-divinylbenzene), polymethacrylate, and silica-based monolithic columns while expanding to polymer-based monoliths with incorporated metal organic frameworks and to vinylized hybrid silica monoliths. In addition, the potential application of porous layer open tubular monolithic columns in low-pressure gas chromatography will be addressed.

Enhanced Separation Capability of Sequential Injection Chromatography for Fluorimetric Determination of Intracellular Dissolved Free Amino Acids in Marine Microalgae.

This chapter describes improvements in a sequential injection method to automate the fluorimetric determination of amino acids by pre-column derivatization with o-phthaldialdehyde in presence of 2-mercaptoethanol. Separation is achieved by reversed-phase liquid chromatography in a 50×4.6mm C18 silica-based monolithic column. The method is low-priced, and the separation is performed by stepwise gradient elution using six mobile phases. The mobile phase used for the first elution step is composed of methanol/tetrahydrofuran/10mM phosphate buffer (pH7.2) at volumetric ratio 8:1:91. Additional elution steps use mobile phases containing methanol and 10mM phosphate buffer at volumetric ratios of 17.5:82.5, 25:75, 35:65, 50:50, and 65:35. Nineteen chromatographic peaks are observed in a mixture of twenty amino acids. The only complete co-elution is between tryptophan and methionine. The entire cycle of amino acid derivatization, chromatographic separation, and column conditioning at the end of separation lasts for 30min. The method is successfully applied to quantify the major intracellular dissolved free amino acids in the marine microalgae Tetraselmis gracilis, Phaeodactium tricornutum, and Synechococcus elongatus.

Comprehensive two-dimensional monolithic liquid chromatography of polar compounds.

Two-dimensional liquid chromatography largely increases the number of separated compounds in a single run, theoretically up to the product of the peaks separated in each dimension on the columns with different selectivities. On-line coupling of a reversed-phase column with an aqueous normal-phase (hydrophilic interaction liquid chromatography) column yields orthogonal systems with high peak capacities. Fast on-line two-dimensional liquid chromatography needs a capillary or micro-bore column providing low-volume effluent fractions transferred to a short efficient second-dimension column for separation at a high mobile phase flow rate. We prepared polymethacrylate zwitterionic monolithic micro-columns in fused silica capillaries with structurally different dimethacrylate cross-linkers. The columns provide dual retention mechanism (hydrophilic interaction and reversed-phase). Setting the mobile phase composition allows adjusting the separation selectivity for various polar substance classes. Coupling on-line an organic polymer monolithic capillary column in the first dimension with a short silica-based monolithic column in the second dimension provides two-dimensional liquid chromatography systems with high peak capacities. The silica monolithic C18 columns provide higher separation efficiency than the particle-packed columns at the flow rates as high as 5mL/min used in the second dimension. Decreasing the diameter of the silica monolithic columns allows using a higher flow rate at the maximum operation pressure and lower fraction volumes transferred from the first, hydrophilic interaction dimension, into the second, reversed-phase mode, avoiding the mobile phase compatibility issues, improving the resolution, increasing the peak capacity, and the peak production rate.

Keeping pace with the increasing demand for high quality drug candidates in pharmaceutical research: Development of a new two-step preparative tandem high performance chromatographic system for the purification of antibodies

In the area of biological drug development, high throughput (HT) technologies are key to identifying the most promising therapeutic candidate in a time-efficient and market-competitive manner. While efficient cloning and expression methods exist, HT downstream processing mainly relies on liquid handling workstations applying miniaturized chromatography columns or resin-based 96-well plates to shorten process development time. However, there is still a lack of generic, preparative chromatographic methods devoid of aggregates and endotoxins with sufficient throughput. The only truly generic antibody purification strategy including an efficient dimer removal consists of Protein A capture followed by size exclusion chromatography (SEC) as a polishing step. Other polishing methods, including IEX, HIC, and CHT, require an antibody-specific fine tuning. However, standard preparative SEC setups tend to be rather time-consuming, and so limit throughput.In this work, we devised a unique chromatography setup enabling an unattended two-step purification of IgGs on the milligram scale directly from 35 mL clarified cell supernatants, processing up to 48 samples in 44.0 h. By introducing a silica-based SEC column, preparative SEC could be accelerated. By further developing an HT two-step preparative Protein A/alternating column regeneration SEC system using Agilent 1260 Infinity LC components, mAbs can be purified generically by two chromatographic steps in 55 min. In this way, by using a 2-position/10-port valve and two quaternary pumps, two SEC columns can be run in parallel, excluding the cleaning and equilibrating phase from the actual cycle time. By further applying a third pump, the Protein A step can be run independently, resulting in a time-optimized process nesting. By introducing a CETAC ASX-520 autosampler, 48 samples can be run automatically without any user intervention over two working days. The developed system is highly reproducible for all tested human IgG1 antibodies, easily generating milligram scale material sufficient for full characterization of the antibodies and for their use in in vitro and in vivo activity assessments.

Silica-based hybrid porous layers to enhance the retention and efficiency of open tubular capillary columns with a 5 μm inner diameter

We report on possibility to enhance the hydrophobicity of octadecylsilylated silica-based porous layered open tubular (PLOT) columns with an inner diameter (i.d.) of 5 μm by applying hybrid tetramethoxysilane (TMOS)/methyltrimethoxysilane (MTMS) layers with inserted methyl groups. Due to this higher hydrophobicity, thinner porous layers suffice to achieve similar retention factor (k) as in octadecylsilylated silica-based PLOT columns synthesized using TMOS only. Since thinner layers have a lower intra-layer mass transfer resistance, this in turn allows to obtain superior column efficiencies in comparison with separations carried out with TMOS-based PLOT columns at the same retention. Since layer thickness contributes to the C-term type of band broadening, this is most pronounced at high velocities. Typical gains in column efficiency at a reduced velocity of νi = 30 are on the order of 15%.Preparing the hybrid PLOT columns in 5 μm i.d.-capillaries with a length of 0.4 m using different TMOS/MTMS preparation mixtures leads to different layer thickness in the capillaries. It is shown that column efficiencies for the most retained compound (k = 0.9–1.5) went from N = 101,000 for PLOT columns with a layer thickness (df) of 250 nm, over N = 95,000 for df = 320 nm to N = 89,000 for df = 400 nm, corresponding to plate heights (H) in the order of 3.5–3.9 μm (reduced plate heights (h = 0.8–1.0)). By applying the same preparation mixtures for much longer capillaries of 1.3 m, a high repeatability of the volumetric phase ratio (m) (difference <1%) and the k-values (difference <5%) was observed between the 0.4 m and 1.3 m PLOT columns. In addition, also a very similar band broadening was obtained, as the h-values in the longer columns coincided well (order of a few % difference) with the reduced plate height curves measured in the shorter columns. The effect of the retention factor and layer thickness on these reduced plate height curves furthermore fits well with the Golay-Aris theory. Depending on the layer thickness, plate numbers in the longer capillary columns were varying from N = 282,000 to N = 379,000 for the most retained compound.