Synthesis Of Stationary Phases Research Articles

Graphene oxide-based stationary phase synthesis and separation of drug molecules for liquid chromatography (HPLC) analysis

A new graphene oxide-based liquid chromatography stationary phase was synthesized, characterized, and used for the first time in the separation of paracetamol and caffeine in pharmaceutical preparations under reversed phase conditions. For this purpose, graphene oxide was synthesized using the modified Hummers method, and Si-GO stationary phase was obtained by immobilizing graphene oxide on aminopropyl silica. The obtained graphene oxide, Si–GO and aminopropyl silica materials were characterized by XRD, SEM, TEM and FTIR analysis. Different mobile phases were used to separate drug molecules in order to determine the mechanism of interaction between drug molecules and the stationary phase. Once the appropriate solvent system was determined, optimum chromatographic parameters such as mobile phase composition (methanol percentage in water), flow rate and column temperature were determined. The linearity of the method was in the range of 0.989–0.997. The maximum relative standard deviation (% RSD) value was calculated as 0.54. Mean recoveries for paracetamol and caffeine were 98%–99%, respectively, and LOD and LOQ were 6 × 10−3 to 6.5 × 10−3 mg/L and 2 × 10−2 to 2.2 × 10−2 mg/L, respectively. The results showed that the new graphene oxide-based stationary phase could be used for the separation of paracetamol and caffeine in pharmaceutical preparations.

Solvent Influence on Zeta Potential of Stationary Phase-Mobile Phase Interface.

Zeta potential is a surface characteristic formed on the solid surface and liquid interface. It is an interesting way to describe the surface properties of materials; thus, a series of four homemade polar embedded stationary phases that contain phosphate groups incorporated into hydrophobic ligands were investigated according to surface zeta potential. Measurements were carried out using Zetasizer Nano ZS for the stationary phases suspensions prepared in various solvent and solvent binary mixtures. The negative zeta potential values were obtained for most cases due to negatively charged residual silanols and phosphate groups. However, in some solvents: tetrahydrofuran, isopropanol, and toluene zeta potential are positive. Additionally, it was observed that the zeta potential seems to be independent of the type of silica gel used for the stationary phase synthesis.

Controllable organosilane monolayer density of surface bonding using silatranes for thiol functionalization of silica particles for liquid chromatography and validation of microanalytical method for elemental composition determination

The present work systematically investigates a new strategy for the functionalization of silica gel using alkyl silatrane chemistry instead of alkylsilanes for synthesis of chromatographic stationary phases. In this work, silica was chemically modified for further functionalization by a thiol-ene click reaction. Thus, 3-mercaptopropylsilatrane (MPS) was used which is capable to form self-assembled monolayers (SAM) on top of silanol surfaces in a controlled manner as previously shown for silicon wafers. The utility of this chemistry for stationary phase synthesis in liquid chromatography was not evaluated yet. Hence, silica surface modifications using MPS were studied in comparison to established 3-mercaptopropyltrimethoxysilane (MPTMS) chemistry. First, the employed elemental analysis method was validated and it showed excellent intra-day and inter-day precisions (typically less than 5% RSD). It could be shown that the reaction kinetics of MPS was roughly 35-times faster than with MPTMS. After 30 min reaction time with MPS, the thiol content reached 74% of the maximal coverage. Due to controlled chemistry with MPS, which does not lead to oligomeric siloxane network at the silica surface, the ligand coverage was lower. However, multiple silanization cycles with MPS led to a dense surface coverage (around 4 µmol m−2). 29Si cross polarization/magic angle spinning (CP/MAS) solid-state NMR revealed distinct T1/T2/T3 ratios for MPS and MPTMS materials with up to 80% T3 (indicative for trifunctional siloxane linkage) for MPS and around 20% T3 for MPTMS. This indicates a more homogeneous, thinner monolayer film of MPS on the silica surface, as compared to an irregular thick oligomeric siloxane network with MPTMS. Bonding of quinine carbamate as chiral selector afforded an efficient chiral stationary phase (CSP) for chromatographic enantiomer separation. Separation factors were comparable to MPTMS-bonded CSP, however, chromatographic efficiency was much better for the MPS-bonded CSP. H/u curves indicated a reduced mass transfer resistance by roughly factor 3 for MPS- compared to MPTMS-bonded CSP. This confirms better chromatographic performance of surfaces with homogeneous monolayer compared to network structures on the silica surface which suffer from poor stationary phase mass transfer.

Synthesis of stationary phases that provide group recognition for polychlorinated biphenyls by porogenic fragment template imprinting.

Molecular recognition based on imprinted polymers results from the polymerization of functional monomers and cross-linkers in the presence of a target analyte (i.e. template), with subsequent removal of the template to create synthetic binding sites. However, complete removal of the template is difficult to achieve, thereby leading to template leaching, which adversely affects real-world analytical applications. To overcome this challenge, the present study utilizes porogenic fragment template imprinting techniques to provide an alternative synthetic strategy to generate molecularly imprinted polymers with molecular recognition toward polychlorinated biphenyls. Thereafter, thus-generated imprinted polymers have been applied as stationary phases in molecularly imprinted solid-phase extraction for preconcentrating six "indicator polychlorinated biphenyls" in both organic and aqueous media. Recoveries of up to 98.9% (imprinted polymers) versus 73.0% (conventional C18 ) in an organic phase, and up to 97.4% (imprinted polymers) versus 89.4% (C18 ) in an aqueous phase have been achieved corroborating the utility of this advanced sorbent material. Finally, porogenic fragment template imprinting strategies have yielded molecularly imprinted polymers that are useful for the quantitative determination of polychlorinated biphenyls in environmental matrices, which provides a low-cost strategy for tailoring stationary phases that avoid template leaching in applications in solid-phase extraction as well as liquid chromatography.

Synthesis of stationary phases containing pyridine, phenol, aniline and morpholine via click chemistry and their characterization and evaluation in supercritical fluid chromatography

Stationary phases containing pyridine, phenol, aniline and morpholine groups were synthesized using copper (I)-catalyzed azide-alkyne cycloaddition click reactions. The backbone of the stationary phases was aminopropyl silica. The stationary phases were evaluated in packed-column supercritical fluid chromatography (pSFC) with acidic and basic solutes without addition of additives. The analysis of metoclopromide and its impurities by SFC-time-of-flight mass spectrometry (SFC-TOFMS) on the phenol phase is presented. In the click reaction, the 1,2,3-triazole ring is formed and to assess its influence on the polarity/selectivity, the click phases were compared to a commercial available 1,2,4-triazole hydrophilic interaction liquid chromatography (HILIC) phase. The phases were also compared to two extensively used stationary phases in SFC namely 2-ethyl pyridine and bare silica. To allow comparison with other phases used in SFC, linear solvation energy relationships (LSER) of the click phases were determined.

A novel ionic-bonded cellulose stationary phase for saccharide separation

A hydrophilic interaction liquid chromatography (HILIC) stationary phase of cellulose-coated silica was synthesized as a novel saccharide separation material. The material, prepared with a method based on ionic interaction, was demonstrated to be efficient for immobilization of saccharides on silica supports. The method is more efficient than traditional immobilized saccharide stationary phase synthesis methods. It was evolved from a method using anion exchanger microparticles agglomerated onto macroparticles of cation exchangers to produce anion exchangers. Cationic cellulose, which has a large number of hydroxyl groups, was immobilized on sulfonated silica. The cellulose-coated stationary phase we designed used strong hydrogen bonding between cellulose hydroxyl and carbohydrate compounds for HILIC retention and separation. The stationary phase was successfully used to separate the samples of polar compounds and the complex samples of oligosaccharides, and demonstrated good reproducibility and stability. The material exhibited good separation selectivity for carbohydrates and ability to enrich glycosylated peptides. The method described here is easy to achieve, environmentally safe and innovative than other methods. It also has extensive application possibilities to separate other categories polar compounds.

Residual silanols at reversed‐phase silica in HPLC – a contribution for a better understanding

As porous silica gel is the most common adsorbent and support for bonded stationary phase synthesis, residual silanol groups are a recurring problem in the field of liquid chromatography and other separation techniques. Residual silanols most often have a negative effect on the separation process by causing peak tailing. Therefore, there was an attempt to remove residual silanols during stationary-bonded phase synthesis. The type and surface concentration of residual silanols were measured using different instrumental techniques such as NMR and infrared spectroscopy, calorimetry, and various chromatographic methods. Residual silanols exhibit acidic characteristic and they can ionize depending on the environment. Thus, they posses cation-exchange properties and cause the zeta potential of silica particle in liquid environment. Presented review discusses the influence of the residual silanol groups on the solvation process and retention of polar compounds. The novel methodology of residual silanols determination is presented as well as the influence of the silanols on the zeta potential of the stationary-bonded phases in chromatographic conditions.

Methodology for porting retention prediction data from old to new columns and from conventional-scale to miniaturised ion chromatography systems

Several procedures are available for simulating and optimising separations in ion chromatography (IC), based on the application of retention models to an extensive database of analyte retention times on a wide range of columns. These procedures are subject to errors arising from batch-to-batch variability in the synthesis of stationary phases, or when using a column having a different diameter to that used when the database was acquired originally. Approaches are described in which the retention database can be recalibrated to accommodate changes in the stationary phase (ion-exchange selectivity coefficient and ion-exchange capacity) or in the column diameter which lead to changes in phase ratio. The entire database can be recalibrated for all analytes on a particular column by performing three isocratic separations with two analyte ions. The retention data so obtained are then used to derive a “porting” equation which is employed to generate the required simulated separation. Accurate prediction of retention times is demonstrated for both anions and cations on 2 mm and 0.4 mm diameter columns under elution conditions which consist of up to five sequential isocratic or linear gradient elution steps. The proposed approach gives average errors in retention time prediction of less than 3% and the correlation coefficient was 0.9849 between predicted and observed retention times for 344 data points comprising 33 anionic or cationic analytes, 5 column internal diameters and 8 complex elution profiles.

Stationary phases for chromatography prepared by ring opening metathesis polymerization

The chemistry of metathesis polymerization-derived stationary phases is summarized. Since both ring-opening metathesis and 1-alkyne polymerization triggered by well-defined transition metal alkylidenes are living polymerization methods, they allow for the controlled and highly reproducible synthesis of stationary phases in terms of both the nature and total content of the functional group(s) of interest. In addition, the high functionality tolerance of these polymerization techniques allows for creating chromatographic supports with an unprecedented diversity in terms of functional groups that may be introduced. Their applications in various areas of separation science such as SPE, ion-chromatography, RP chromatography, chiral chromatography, and the high-performance liquid chromatographic separation of biomolecules are summarized. Within the context of the latter topic, special attention will be given to metathesis polymerization-derived monolithic supports. Consideration will also be given to those aspects of polymer chemistry that are relevant to the separation performance of these supports.

Evaluation of Polymeric Methacrylate-based Monoliths in Capillary Electrochromatography for their Potential to Separate Pharmaceutical Compounds

Polymeric methacrylate-based monoliths are evaluated in capillary electrochromatography (CEC) and pressurized capillary electrochromatography (p-CEC) for their potential in pharmaceutical analysis. Using a given polymerization mixture as a basis for the monolith synthesis, different mobile phase pH at constant organic modifier concentrations are tested in both CEC and p-CEC. The test set consists of basic, acidic, amphoteric, and neutral compounds, which are mainly pharmaceuticals. Because of the mainly hydrophobic character of the stationary phase, the interactions are largest when the compounds appear in an uncharged state, but some ion-exchange phenomena with negatively charged compounds can also be observed. In CEC, acidic substances are most retained at low pH. For amphoteric and neutral compounds, no preference regarding analyzing pH can be derived from these experiments. For basics, a high pH is chosen, but a reduced solvent strength is needed to enhance the retention of these compounds. The retention mechanism in p-CEC can also be assigned to both hydrophobic and ionic interactions. For acidic, amphoteric, and neutral compounds, acceptable retention is seen. For the basic compounds, the retention with a mobile phase containing 50% organic modifier is low, as in CEC. However, when the organic modifier content in the mobile phase is decreased, retention increases and the selectivity of the stationary phase is more pronounced. This mode of operation presents a possibility for separating some test mixtures, thus some potential for pharmaceutical analysis is seen. More efforts are needed to obtain higher efficiencies and better peak shapes, which might be solved by a further optimization of both the stationary phase synthesis and the mobile phase composition.

Thermodynamic equilibrium of the solute distribution in size-exclusion chromatography

Our understanding of the nature of solute retention in size-exclusion chromatography (SEC) is predicated upon an equilibrium, entropy-controlled, size-exclusion mechanism. The entropic nature of the separation depends, in turn, upon the solute distribution coefficient ( K SEC) being at (or close to) thermodynamic equilibrium. Classic experiments to confirm this assumption were performed over thirty years ago. Here, we combine information obtained from both flow and static mixing SEC experiments to show that the solute distribution in SEC is in thermodynamic equilibrium over a molar mass range extending one order of magnitude higher than previously measured (from 2×10 3 to 1.1×10 6 Da) using crosslinked polystyrene packing material of identical pore size (10 4 Å). The differences between our observations and previous ones conducted over three decades ago are ascribed, principally, to advances in stationary phase synthesis and column technology for SEC in particular and, secondarily, to improvements in the performance of the various instrumental components of liquid chromatographic systems in general.

Studies of physicochemical properties of the surfaces with the chemically bonded phase of BSA

The paper presents the method of preparation and determination of physicochemical properties of silica gels with a chemically bonded BSA phase as well as studies of the effect of support porosity on the synthesis. Wide-porous Z-300 and narrow-porous Z-100 silica gels were studied. The investigations showed a significant effect of pore size on the synthesis of stationary phases with BSA. Modification with protein results in changes of adsorption properties and porosity of adsorbent samples. Changes of physicochemical properties result in significant changes of geometrical and structural heterogeneity of the support (specific surface area, fractal coefficient) as well as energetic heterogeneity of the samples.

Dynamic control of Dps protein levels by ClpXP and ClpAP proteases in Escherichia coli.

The Escherichia coli starvation-induced DNA protection protein Dps was observed to be degraded rapidly during exponential growth. This turnover is dependent on the clpP and clpX genes. The clpA gene is not required for Dps proteolysis, suggesting that Dps is a substrate for ClpXP protease but not for ClpAP protease. Dps proteolysis was found to be highly regulated. Upon carbon starvation, Dps is stabilized, which together with increased Dps synthesis allows strong accumulation of Dps in the stationary phase. The addition of glucose to starving cells results in rapid resumption of Dps proteolysis by ClpXP. Oxidative stress also leads to efficient stabilization of Dps. After hyperosmotic shift, however, proteolysis remains unaffected. Thus, regulated proteolysis of Dps strongly contributes to controlling Dps levels under very specific stress conditions. In contrast to the regulated degradation of RpoS by ClpXP, Dps proteolysis is independent of the recognition factor RssB. In addition, during starvation, clpP and, to a somewhat lesser extent, clpA are involved in maintaining ongoing Dps synthesis (acting at the level of Dps translation), which is required for strong Dps accumulation in long-term stationary phase cells. In summary, both ClpXP and ClpAP exert significant control of Dps levels by affecting log phase stability and stationary phase synthesis of Dps respectively.

A new approach to human serum albumin chiral stationary phase synthesis and its use in capillary liquid chromatography and capillary electrochromatography

Continuous beds with immobilised human serum albumin as a chiral selector were synthesised in fused-silica capillaries in a modified fashion using additives during the protein allylation and polymerisation steps. Acetyl salicylic acid and L-tryptophan were used as additives to interact with the active centres that are responsible for chiral recognition, thus preserving them during the protein coupling procedure. The beds synthesised under modified conditions exhibit higher resolutions and enantioselectivities in capillary liquid chromatography, especially when L-tryptophan was used as an additive. Monolithic adsorbent synthesised with L-tryptophan possesses much higher efficiency when capillary electrochromatography was employed.

Performance of capillary columns for high-temperature gas chromatography

The developments in stationary-phase synthesis and capillary column technology have opened new perspectives in the analysis of high-molecular-weight compounds (600 daltons) and thermolabile organic compounds by high-temperature-high-resolution gas chromatography. This branch of high-resolution gas chromatography deals with analysis performed up to 390 degrees C oven temperature (with some applications going up to 420 degrees C and even a few applications to 450 degrees C maximum). The technique has been applied to many different fields of science (e.g., organic geochemistry, environmental chemistry, archeology, and natural product research). Apolar and medium-polar gum phases can now be operated at temperatures from 400 to 480 degrees C, but these higher temperatures are seldom used because of the thermostability of the material used to make the capillary tubing. This paper shows the performance of nine commercial high-temperature columns when used in routine applications.

Estado da arte da cromatografia gasosa de alta resolução e alta temperatura

The developments in stationary phase synthesis and capillary column technology, have opened new perspectives in analysis of high molecular mass compounds (³600 daltons) and thermolabile organic compounds by High Temperature High Resolution Gas Chromatography (HT-HRGC). HT-HRGC is a new analytical borderline and its application to the analysis of high molecular mass compounds is still in its infancy. The apolar and medium polar gum phases can now be operated at temperatures up to 400-480oC, being used for the analysis of n-alcanes up to C-100, lipids, oligosaccharides, industrial resins, polyglycerols, cyclodextrins, porphyrins, etc. This technique should play a leading role as a powerful tool, for many different analysis types, in multidisciplinary fields of Science.

A Methodology for the Characterization of Ion-Exchange Resins

Tremendous strides have been made in the field of stationary phase synthesis over the course of the last decade. Although important research has been carried out to elucidate the characteristics of various resins, there is currently a lack of understanding regarding the effect of the various resin materials on preparative modes of chromatography. To describe preparative chromatography, one needs to have appropriate isotherm and transport models. In this article a methodology is presented to enable the identification of appropriate transport models to describe the chromatographic behavior of solutes in preparative ion-exchange systems. The methodology involves simple pulse experiments to estimate the various transport parameters followed by the construction and analysis of various dimensionless groups to identify the dominant transport mechanisms in a given resin. Following this, one can identify an appropriate transport model to describe the chromatographic behavior of solutes on the resin material. This model is then employed in concert with the steric mass action (SMA) isotherm and is validated using experimental data. The results presented provide significant insight into the identification of dominant transport mechanisms on various ion-exchange resin systems.

High-temperature high-resolution gas chromatography: breaching the barrier to the analysis of polar and high molecular weight compounds

Developments in stationary phase synthesis and capillary column technology have opened new perspectives in the analysis of high molecular weight compounds (≥600 Da) and thermolabile organic compounds by high-temperature high-resolution gas chromatography (HT-HRGC). Apolar and medium polarity gum phases can now be operated at temperatures up to 400–480°C, and are being used for the analysis of compounds including n-alkanes up to C 100, lipids, oligosaccharides, industrial resins, polyglycerols, cyclodextrins, and porphyrins.

Electrospray Liquid Chromatography/Mass Spectrometry of Ginsenosides

An electrospray liquid chromatography/mass spectrometry (LC/MS) method has been developed for the analysis of ginseng saponins (ginsenosides) contained in extracts of the root of Panax ginseng (Korean ginseng) and Panax quinquefolius (American ginseng). The LC/MS method consists of separation of ginsenosides using an (aminopropyl)silica HPLC column, followed by detection using a photodiode array UV absorbance detector and then on-line electrospray mass spectrometry. Ginsenosides eluted from the HPLC column in order of increasing molecular weight and were detected as [M + Na] + or [M + 138] + adducts. Occurrence of [M + 138] + adducts was most prominent when new HPLC columns were used, and none were observed when solutions of ginsenoside standards were infused into the mass spectrometer without use of HPLC. The cation weighing 138 was identified as (3-ammoniumpropyl)trihydroxysilane, + NH 4 (CH 2 ) 3 Si(OH) 3 , which was either a byproduct of stationary phase synthesis or a stationary phase degradation product. LC/MS chromatograms of extracts of Korean ginseng and American ginseng demonstrated substantial differences between the relative amounts of each ginsenoside. Based on molecular weight and coelution with standards, ginsenosides R g1 , R e , R d , R c , R b2 , and R b1 (in order of elution from the HPLC column) were identified in both ginseng extracts. Four other ginsenosides were detected by mass spectrometry for which no standards were available, and their molecular weights were 801 (possibly corresponding to ginsenoside R f ), 817, 947, and 963 (possibly 20-gluco-R f ). The ginsenosides weighing 817 and 963 were detected only in the Korean ginseng extract.

Synthesis of siloxane stationary phases for capillary gas chromatography and supercritical fluid chromatography

AbstractA comparison is made between dichlorosilanes and cyclic siloxanes as starting materials in the synthesis of stationary phases for capillary gas chromatography (CGC) and supercritical fluid chromatography (SFC). Siloxanes containing one or more of the side groups methyl, vinyl, phenyl, and cyanoethyl in various ratios were synthesized and compared. These phases were characterized by chromatographic (gel permeation, GPC), spectroscopic (IR, 1H NMR, 29Si NMR), and thermal (DSC) methods. Coated fused silica columns were evaluated with respect to polarity, crosslinkability with several free‐radical initiators, and thermal stability. A new liquid phase, 7% cyanoethyl, 7% phenyl, 1% vinyl methyl polysiloxane is shown to be more polar than OV‐1701, more temperature stable, easily crosslinked and suitable for use in supercritical fluid chromatography.