Chromatographic Stationary Phases Research Articles

Electrochemically Modulated Liquid Chromatography in Fused Silica Capillary Columns.

Electrochemically modulated liquid chromatography (EMLC) uses electrical potentials, applied to a conductive chromatographic stationary phase (e.g., porous graphitic carbon [PGC]), to manipulate analyte retention. This paper reports the design of a capillary EMLC column with a smaller internal diameter (ID; 250 μm) than that of the standard bore predecessor (3.3 mm ID). The new capillary EMLC columns are configured so that the PGC stationary phase serves as the working electrode in a two-electrode electrochemical cell and simplifies electrode placement by obviating the need for a counter electrode. This configuration also eliminates the internal Nafion sleeve that is critical to operation for the standard bore columns, thereby avoiding Nafion deformation as a source of chromatographic band broadening and rupturing as a mode of column failure. Indeed, values for chromatographic efficiency obtained on the capillary columns meet or exceed those measured for the standard columns (20 000-40 000 vs 14 000 plates/m, respectively) with near symmetric elution bands (asymmetry factors of 1.1-1.4 for well-packed capillaries) that surpass band symmetries observed in all prior studies. A test suite of aromatic sulfonates was used to characterize the chromatographic performance of the capillary EMLC columns. Separations of this test mixture showed that retention factors for individual analytes could be manipulated by as much as 21× by changing the applied potential at the PGC stationary phase. Changes in retention behavior at different potential ranges, hypothesized to result from differences in adsorption orientation, were also observed and are consistent with past work. Collectively, the retention behavior unique to EMLC is operative in this new capillary configuration and promises to open new avenues in tuning LC separations.

Preparation and evaluation of hydrophobically associating polyacrylamide coated silica composite as high performance liquid chromatographic stationary phase

A hydrophobically associating polyacrylamide (HAPM)-coated silica packing (HAPM@silica) as a novel stationary phase was synthesized by a simple thermal immobilization to separate multiple polar compounds for the first time. The successful immobilization of HAPM on the silica support was confirmed by elemental analysis, scanning electron microscopy (SEM) and the Brunauer–Emmett–Teller (BET). The thickness of HAPM coating is crucial and the one-layer HAPM@silica stationary phase was demonstrated the highest separation efficiency. Compared with the bare silica and amino modified silica, the new stationary phase showed better separation performance for multiple polar compounds such as bases, alkaloids, carbohydrates and amino acid compounds under the experimental conditions. The chromatographic behaviors were evaluated by investigating the effects of acetonitrile content, water content, mobile phase pH on separation. A typical hydrophilic interaction liquid chromatography (HILIC) retention feature of HAPM based column was observed at the low percentage of water content. The packing material also showed excellent chromatographic repeatability with intraday RSDs of the retention time of five bases less than 0.46% (n = 10) and interday RSDs less than 1.45% (n = 5). In summary, the new chromatographic material can be used for a feasible option for analysis of polar compounds.

Development and Analytical Validation of the Methodology for Vitamins in Tablets by Ultra-Performance Liquid Chromatography.

In the present study, we developed a reliable and robust chromatographic method for the quantification of multivitamins in tablet samples by ultra-performance liquid chromatography (UPLC) with photodiode array detection. The vitamins nicotinamide, pyridoxine, riboflavin, and thiamin were analyzed and quantified in a total analysis time of 2.5minutes, using hydrophilic interaction liquid chromatography stationary phase. Tocopherol acetate and cyanocobalamin were analyzed and quantified in a total analysis time of 2.5minutes, using reversed-phase (RP)-UPLC. The analysis time reported here is lower than that of similar methods reported in the literature for single vitamin determination. The method linearity exhibits a good correlation coefficient (R2=0.998) with the relative residual standard deviation in the acceptable limit of 2.0%. The developed methods were validated, and the results demonstrated that the proposed analytical method showed to be selective, sensitive, accurate, and robust for the quantification of evaluated vitamins in multivitamin tablets. The work was fully developed in the quality control laboratory of a pharmaceutical industry in the Agroindustrial District of Anápolis (DAIA, Goiás, Brazil), where the product is manufactured.

Fast separation of water-soluble vitamins by hydrophilic interaction liquid chromatography based on submicrometer flow-through silica microspheres

In the present study, submicrometer flow-through silica microspheres (Sub-FTSiO2) was for the first time obtained via a suspension polymerization method coupled with sol-gel transition and phase separation. The Sub-FTSiO2 was characteristic of rich mesopores, penetrable macropores and small particle size, which would be beneficial to fast mass transfer, low column backpressure and high column efficiency. It was directly used as the hydrophilic interaction liquid chromatographic (HILIC) stationary phase, and the fast separation of seven water-soluble vitamins in 2.2 min was realized. The proposed method was successfully applied to the determination of water-soluble vitamins in two functional beverages on the market. The prepared Sub-FTSiO2 was well demonstrated for fast HILIC, and would be potential as the stationary phase matrix for fast liquid chromatography in diverse separation modes.

Camptothecin-imprinted polymer microspheres with rosin-based cross-linker for separation of camptothecin from Camptotheca acuminata fruit

In this work, camptothecin (CPT) molecularly imprinted polymer (MIP) microspheres were prepared via membrane emulsification-suspension polymerization using ethylene glycol maleic rosinate acrylate as a crosslinker, acrylic acid as a functional monomer, and CPT as a template molecule. The MIPs were characterized by scanning electron microscopy, laser granulometry, nitrogen sorption porosimetry, and thermogravimetric analysis. The results indicated that the MIPs had a regular spherical shape with porous surfaces and particle sizes mainly ranging from 3 to 5 μm. The surface areas of the MIPs and non-molecularly imprinted polymer (NIP) microspheres were 89.44 and 86.91 m2 g−1, respectively, and their respective average pore sizes were 11.42 and 10.84 nm. Furthermore, the MIPs and NIPs exhibited excellent thermal stabilities. The HPLC columns prepared with the MIPs and NIPs as the chromatographic stationary phase showed good fluidity. The effect of the MIP column on CPT was evaluated with HPLC, and the selectivity and separation performance of the MIP and NIP columns for CPT and 10-hydroxycamptothecin were evaluated. The chromatographic thermodynamics and separation mechanism were also studied. Finally, the MIP column was successfully applied to the separation and purification of CPT from an extract of Camptotheca acuminata fruit, and the reusability of the MIP column was also investigated.

Matrix-assisted DOSY

The analysis of mixtures by NMR spectroscopy is challenging. Diffusion-ordered NMR spectroscopy enables a pseudo-separation of species based on differences in their translational diffusion coefficients. Under the right circumstances, this is a powerful technique; however, when molecules diffuse at similar rates separation in the diffusion dimension can be poor. In addition, spectral overlap also limits resolution and can make interpretation challenging. Matrix-assisted diffusion NMR seeks to improve resolution in the diffusion dimension by utilising the differential interaction of components in the mixture with an additive to the solvent. Tuning these matrix-analyte interactions allows the diffusion resolution to be optimised. This review presents the background to matrix-assisted diffusion experiments, surveys the wide range of matrices employed, including chromatographic stationary phases, surfactants and polymers, and demonstrates the current state of the art.

Polyacrylamide-alginate (PAAm-Alg) and phospho-L-tyrosine-linked PAAm-Alg monolithic cryogels: Purification of IgG from human serum

In recent decades cryogels as monolithic materials have gained interest as stationary phase in chromatography for purification of biomolecules. In this study, polyacrylamide-alginate (PAAm-Alg) monolithic cryogels were prepared by cryo-copolymerization of acrylamide and alginate monomers and methylene-bisacrylamide as crosslinker to be used as a matrix in affinity chromatography for purification of proteins. Ortho-phospho-L-tyrosine (P-Tyr) was covalently attached onto PAAm-Alg cryogels via bisoxirane-activation (PAAm-Alg-Bix-P-Tyr) and both derivatized and non-derivatized cryogels were utilized for the purification of immunoglobulin G (IgG) from human serum. Cryogels were characterized by scanning electron microscopy, swelling tests, elemental analysis, FTIR, and flow dynamics. The effects of buffer systems, conductivity, and pH on IgG adsorption were studied. Through breakthrough curve analysis a dynamic capacity of 9.2 mg IgG/mL with an IgG purity of 94% was obtained (based on ELISA analysis of IgG and albumin) for PAAm-Alg-Bix-P-Tyr cryogel when human serum was diluted in 10 mmol/L NaP buffer at pH 6.0. The adsorption isotherm data were well described by the Langmuir model with value of maximum adsorption capacity of 36.12 ± 3.63 mg of IgG/g for PAAm-Alg-Bix-P-Tyr. The PAAm-Alg-Bix-P-Tyr cryogel provides an attractive alternative for adsorption of IgG from human serum.

Characterization of three macrocyclic glycopeptide stationary phases in supercritical fluid chromatography

Macrocyclic glycopeptides have been used as chromatographic stationary phases for over twenty years, particularly for their ability to separate enantiomers. While they are mostly used with buffered aqueous liquid mobile phases, they can also be used in supercritical fluid chromatography (SFC) with mobile phases comprising pressurized carbon dioxide and a co-solvent (like methanol), possibly comprising acidic or basic additives.In the present study, we compared three macrocyclic glycopeptide stationary phases (Chirobiotic V2, Chirobiotic T and Chirobiotic TAG) in SFC with carbon dioxide – methanol (90:10) containing no additives. First, the interactions contributing to retention are evaluated with a modified version of the solvation parameter model, comprising five Abraham descriptors (E, S, A, B, V) and two additional descriptors to take account of interactions with ionizable species (D− and D+). Linear solvation energy relationships (LSER) are established based on the retention of 145 achiral analytes.Secondly, the contributions of interactions to enantioseparations are discussed, based on the analysis of 67 racemates. The individual success rate on each phase was observed to be moderate, especially as these phases are known to be more efficient when acidic or basic additives are employed. Chirobiotic TAG proved more successful than the other two phases. Discriminant analyses were computed to gain some insight on retention mechanisms, but only Chirobiotic TAG provided interpretable results.Finally, the effects of a small proportion of acidic or basic additive on enantioseparation with Chirobiotic T stationary phase are briefly discussed.

Ultra-high thermal stability perarylated ionic liquids as gas chromatographic stationary phases for the selective separation of polyaromatic hydrocarbons and polychlorinated biphenyls

Ionic liquids (ILs) are well-known in the field of separation science for their unique selectivity when used as stationary phases in gas chromatography (GC). While a significant amount of knowledge has been attained in correlating structural features of an IL to separation selectivity, developments in producing IL-based stationary phases suitable for high temperature GC studies have lagged behind. Column bleed is a result of the stationary phase undergoing volatilization/decomposition at high temperatures and is undesirable in separations coupled to GC/MS. It has been well-known that traditional classes of ILs with long alkyl side chain substituents are susceptible to Hofmann elimination at elevated temperatures. In this study, a new class of IL stationary phases containing perarylated cations exhibiting improved thermal stability are introduced. These ILs were used to prepare wall-coated open tubular columns with high column efficiency and produced very low bleed at temperatures up to 350 °C. Their unique chemical structures provide stronger π-π interactions compared to many commercially-available stationary phases. To exploit the unique interactions provided by these stationary phases, the separation of two classes of environmentally hazardous aromatic compounds, namely, polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs), was examined. Both classes of compounds contain structural isomers with high boiling points that are often challenging to separate. The perarylated sulfonium and phosphonium IL-based stationary phases exhibited excellent thermal stability as well as unique selectivity toward isomers of PAHs as well as toxic PCB analyte pairs.

Comparison of underivatized silica and zwitterionic sulfobetaine hydrophilic interaction liquid chromatography stationary phases for global metabolomics of human plasma

To increase metabolome coverage in global LC–MS metabolomics, often both reversed-phase liquid chromatography (RPLC) and hydrophilic-interaction liquid chromatography (HILIC) are implemented in parallel. However, there is a lack of consensus in the literature on the best HILIC stationary phase to employ for global metabolomics of human biological fluids. The objective of this study was to compare in detail the performance of two commonly employed HILIC phases: zwitterionic sulfobetaine ZIC-HILIC stationary phase and an underivatized silica HILIC stationary phase. During method development, the effect of salt concentration in the mobile phase was also investigated, and 5 mM ammonium acetate was selected. The stationary phases were evaluated using a mixture of 37 polar standards covering a range of logP values (-10 to 3.73), molecular weights (59–776 Da), charges (15 anions, 11 cations, and 11 neutral) as well as 17 lipid standards to understand phospholipid behaviour on the two stationary phases. The criteria used for the comparison included the quality of the chromatographic peak shape, adequate analyte retention, peak separation capability, and metabolite coverage. The zwitterionic ZIC-HILIC column provided better chromatographic performance over the silica stationary phase with 14 standards achieving good quality peaks compared to the 7 with the silica column. Only 2 standards were undetected with the ZIC-HILIC column compared to the 14 undetected with the silica column. In human plasma, 1966 and 1650 metabolites were observed on the ZIC-HILIC column in positive and negative electrospray ionization (ESI) respectively. On the silica HILIC column, 1773 and 2028 metabolites were observed in positive and negative ESI respectively, showing comparable performance of the two phases. Next, the effect of adding 10 mM ammonium phosphate to the samples to improve the analyte peak shape and metabolite coverage was investigated for both ZIC-HILIC and silica HILIC. In contrast with recently reported results for pZIC-HILIC, there was no clear evidence that ammonium phosphate addition was beneficial for human plasma samples. In conclusion, ZIC-HILIC provided better chromatographic performance for polar plasma metabolomics than underivatized silica in terms of chromatographic peak shape and chromatographic resolution, while maintaining comparable metabolite coverage. The addition of ammonium phosphate to human plasma was not beneficial for either of the two stationary phases.

Front Cover: Comparative study of different chemistries and particle properties, high‐performance liquid chromatography stationary phases in separation of escitalopram oxalate and its impurities in different pharmaceutical dosage forms

Front Cover: Comparative study of different chemistries and particle properties, high‐performance liquid chromatography stationary phases in separation of escitalopram oxalate and its impurities in different pharmaceutical dosage forms

Combination of Three Functionalized Temperature-Sensitive Chromatographic Materials for Serum Protein Analysis.

We have developed a methodology to capture acidic proteins, alkaline proteins, and glycoproteins separately in mouse serum using a combination of three functionalized temperature-responsive chromatographic stationary phases. The temperature-responsive polymer poly(N-isopropylacrylamide) was attached to the stationary phase, silica. The three temperature-responsive chromatographic stationary phase materials were prepared by reversible addition–fragmentation chain transfer polymerization. Alkaline, acidic, and boric acid functional groups were introduced to capture acidic proteins, alkaline proteins, and glycoproteins, respectively. The protein enrichment and release properties of the materials were examined using the acidic protein, bovine serum albumin; the alkaline protein, protamine; and the glycoprotein, horseradish peroxidase. Finally, the three materials were used to analyze mouse serum. Without switching the mobile phase, the capture and separation of mouse serum was achieved by the combination of three temperature-responsive chromatographic stationary phase materials. On the whole, 313 proteins were identified successfully. The number of different proteins identified using the new method was 1.46 times greater than the number of proteins that has been identified without applying this method. To our knowledge, this method is the first combinatorial use of three functionalized temperature-responsive chromatographic stationary phase silica materials to separate proteins in mouse serum.

On the ability of chromatographic mass balance to predict solute diffusivity in drug delivery systems

AbstractThe ability to predict the drug diffusion coefficient within hydrogel‐based drug delivery devices has a pivotal role in the design of these materials. In the last years, many mathematical models have been developed, but they often rely on fitted parameters with a consequent limitation in terms of prediction. Indeed, they are mainly centered on the pure Fickian diffusion together with degradation and swelling contributions. However, especially with a drug concentration typical of pharmacological treatments, several other mechanisms such as drug–polymer and drug–drug interactions cannot be neglected. In this work, we checked the ability of a simple mathematical model to estimate diffusion coefficients of drugs loaded within hydrogel considered as a chromatographic stationary phase. Mathematical modeling satisfactorily matched with different sets of literature data proving that our assumptions are able to describe the key phenomena governing the device's behavior.

Recent Trends in Fast Liquid Chromatography for Pharmaceutical Analysis

Background: Liquid chromatography is the workhorse of analytical laboratories of pharmaceutical companies for analysis of bulk drug materials, intermediates, drug products, impurities and degradation products. This efficient technique is impeded by its long and tedious analysis procedures. Continuous efforts of scientists to reduce the analysis time resulted in the development of three different approaches namely, HTLC, chromatography using monolithic columns and UHPLC. Methods: Modern column technology and advances in chromatographic stationary phase including silica-based monolithic columns and reduction in particle and column size (UHPLC) have not only revolutionized the separation power of chromatographic analysis but also have remarkably reduced the analysis time. Automated ultra high-performance chromatographic systems equipped with state-ofthe- art software and detection systems have now spawned a new field of analysis, termed as Fast Liquid Chromatography (FLC). The chromatographic approaches that can be included in FLC are hightemperature liquid chromatography, chromatography using monolithic column, and ultrahigh performance liquid chromatography. Results: This review summarizes the progress of FLC in pharmaceutical analysis during the period from year 2008 to 2017 focusing on detecting pharmaceutical drugs in various matrices, characterizing active compounds of natural products, and drug metabolites. High temperature, change in the mobile phase, use of monolithic columns, new non-porous, semi-porous and fully porous reduced particle size of/less than 3μm packed columns technology with high-pressure pumps have been extensively studied and successively applied to real samples. These factors revolutionized the fast high-performance separations. Conclusion: Taking into account the recent development in fast liquid chromatography approaches, future trends can be clearly predicated. UHPLC must be the most popular approach followed by the use of monolithic columns. Use of high temperatures during analysis is not a feasible approach especially for pharmaceutical analysis due to thermosensitive nature of analytes.

Epoxide-derived mixed-mode chromatographic stationary phases for separation of active substances in fixed-dose combination drugs.

A method for the preparation of novel mixed-mode reversed-phase/strong cation exchange stationary phase for the separation of fixed-dose combination drugs has been developed. An epoxysilane bonded silica prepared by vapor phase deposition was used as a starting material to produce diol, octadecyl, sulfonate, and mixed octadecyl/sulfonate groups bonded silica phases. The chemical structure and surface coverage of the functional groups on these synthesized phases were confirmed by fourier-transform infrared and solid-state 13 C NMR spectroscopy and elemental analysis. Alkylbenzene homologs, basic drugs, nucleobases and alkylaniline homologs were used as probes to demonstrate the reversed-phase, ion exchange, hydrophilic interaction and mixed-mode retention behaviors of these stationary phases. The octadecyl/sulfonate bonded silica exhibits pronounced mixed-mode retention behavior and superior retentivity and selectivity for alkylaniline homologs. The mixed-mode retention is affected by either ionic or solvent strength in the mobile phase, permiting optimization of a separation by fine tuning these parameters. The mixed-mode stationary phase was applied to separate two fixed-dose combination drugs: compound reserpine tablets and compound methoxyphenamine capsules. The results show that simultaneous separation of multiple substances in the compound dosage can be achieved on the mixed-mode phase, which makes multi-cycles of analysis for multiple components obsolete.

Efficient preparative separation of 6-(4-aminophenyl)-5-methyl-4, 5-dihydro-3(2H)-pyridazinone enantiomers on polysaccharide-based stationary phases in polar organic solvent chromatography and supercritical fluid chromatography.

6-(4-Aminophenyl)-5-methyl-4,5-dihydro-3(2H)-pyridazinone is a key synthetic intermediate for cardiotonic agent levosimendan. Very few studies address the use of chiral stationary phases in chromatography for the enantioseparation of this intermediate. This study presents two efficient preparative methods for the isolation of (R)(-)-6-(4-aminophenyl)-5-methyl-4,5-dihydro-3(2H)-pyridazinone in polar organic solvent chromatography and supercritical fluid chromatography using polysaccharide-based chiral stationary phases and volatile organic mobile phases without additives in isocratic mode. Under optimum conditions, Chiralcel OJ column showed the best performance (α=1.71, Rs=5.47) in polar organic solvent chromatography, while Chiralpak AS column exhibited remarkable separations (α=1.81 and Rs=6.51) in supercritical fluid chromatography with an opposite enantiomer elution order. Considering the sample solubility, runtime and solvent cost, the preparations were carried out on Chiralcel OJ column and Chiralpak AS column (250× 20mm i.d.; 10µm) in polar organic mode and supercritical fluid chromatography mode with methanol and CO2 /methanol as mobile phases, respectively. By utilizing the advantages of chromatographic techniques and polysaccharide-based chiral stationary phases, this work provides two methods for the fast and economic preparation of (R)(-)-6-(4-aminophenyl)-5-methyl-4,5-dihydro-3(2H)-pyridazinone, which are suitable for the pharmaceutical industry.

Refinement strategy for antivenom preparation of high yield and quality.

Antivenoms from hyperimmune animal plasma are the only specific pharmaceuticals against snakebites. The improvement of downstream processing strategies is of great interest, not only in terms of purity profile, but also from yield-to-cost perspective and rational use of plasma of animal origin. We report on development of an efficient refinement strategy for F(ab')2-based antivenom preparation. Process design was driven by the imperative to keep the active principle constantly in solution as a precautionary measure to preserve stability of its conformation (precipitation of active principle or its adsorption to chromatographic stationary phase has been completely avoided). IgG was extracted from hyperimmune horse plasma by 2% (V/V) caprylic acid, depleted from traces of precipitating agent and digested by pepsin. Balance between incomplete IgG fraction breakdown, F(ab')2 over-digestion and loss of the active principle's protective efficacy was achieved by adjusting pepsin to substrate ratio at the value of 4:300 (w/w), setting pH to 3.2 and incubation period to 1.5 h. Final polishing was accomplished by a combination of diafiltration and flow-through chromatography. Developed manufacturing strategy gave 100% pure and aggregate-free F(ab')2 preparation, as shown by size-exclusion HPLC and confirmed by MS/MS. The overall yield of 75% or higher compares favorably to others so far reported. This optimised procedure looks also promising for large-scale production of therapeutic antivenoms, since high yield of the active drug and fulfillment of the regulatory demand considering purity was achieved. The recovery of the active substance was precisely determined in each purification step enabling accurate estimation of the process cost-effectiveness.

Examining the unique retention behavior of volatile carboxylic acids in gas chromatography using zwitterionic liquid stationary phases

For the first time, gas chromatographic (GC) stationary phases consisting of zwitterionic liquids (ZILs) possessing sulfonate functional groups were utilized for the analysis of volatile carboxylic acids (VCAs). The highly polar and acidic nature of VCAs significantly limits the number of currently available GC stationary phases, which are all largely based on acid-modified polyethylene glycol. In this study, it is shown that this class of ZILs exhibit strong retention of VCAs with excellent peak symmetry. Unique chromatographic selectivity toward VCAs is also demonstrated by tuning the structural features of the ZILs. The solvation properties of the three ZILs as well as a structurally similar conventional monocationic IL were characterized using the Abraham solvation parameter model.

Preparation and Chromatographic Evaluation of a L -Isoleucine-Bonded Chromatographic Stationary Phase

Preparation and Chromatographic Evaluation of a L -Isoleucine-Bonded Chromatographic Stationary Phase

Comparative study of different chemistries and particle properties, high‐performance liquid chromatography stationary phases in separation of escitalopram oxalate and its impurities in different pharmaceutical dosage forms

AbstractThe liquid chromatographic separation performance of 12 different columns was evaluated for the estimation of the escitalopram oxalate and six of its impurities in different pharmaceutical formulations. All columns exhibited a reasonable separation power. Six columns with three different chemistries and two different particle morphology (i.e. core‐shell and fully porous particle) were nominated for further evaluation. The objectives of the current study were to develop a simple method for detection of escitalopram oxalate and its impurities and to compare the separation power of the stationary phase of different chemistry and particle properties. The separation was carried out under isocratic elution by using acetonitrile/methanol/water/phosphate buffer solution (pH = 3.5, 50 mM) (25:5:20:50, v/v/v/v) mixture as mobile phase, with a flow rate of 1.2 mL min−1. The core‐shell phenyl‐hexyl column showed the best separation performance over the traditional octadecyl and pentafluorophenyl phases. The developed method was applied for detection of escitalopram oxalate and its impurities in valid and expired dosage forms.