HPLC separation of dihydropyridine derivatives enantiomers with emphasis on elution order using polysaccharide‐based chiral columns

The separation of enantiomers of five chiral dihydropyridine derivatives was studied on five different polysaccharide‐based chiral HPLC columns with various normal‐phase (NP), polar organic, and reversed‐phase eluents. Along with the successful separation of analyte enantiomers, the emphasis of this study was on enantiomer elution order (EEO) with various columns and mobile phase composition. The interesting phenomenon of reversal of EEO, recently reported in the case of amlodipine (AML) depending on the concentration of formic acid in acetonitrile, was also confirmed with NP eluents. Under RP conditions at relatively low water content, the EEO of AML could also be reverted by varying the concentration of formic acid in the mobile phase. However, at higher water content the same parameter did not affect the EEO, but only induced gradual decrease in resolution up to complete co‐elution of enantiomers. Additionally, in organic‐aqueous mobile phases retention factors decreased with increasing water content but only up to 20% (v/v), while above this concentration the expected typical RP behavior was observed. The presence of the commonly used additive diethylamine in the mobile phase seems important for observing a reversal in EEO with increasing concentration of formic acid. The reversal of the EEO was characteristic of AML only and was not observed for any of other dihydropyridines included in this study.     

Enantiomer elution order reversal of fluorenylmethoxycarbonyl-isoleucine in high-performance liquid chromatography by changing the mobile phase temperature and composition

In this paper the elution order reversal of enantiomers of fluorenylmethoxycarbonyl- or FMOC-isoleucine is described depending on the separation temperature and composition of the mobile phase when using the polysaccharide-based chiral column Lux Cellulose-1 in HPLC with normal-phase eluent. Reversal of the enantiomer elution order (EEO) in HPLC depending on the column temperature and content of the polar modifier in the mobile phase has been reported before in the literature. However, EEO reversal by changing the content of acidic modifier in the mobile phase seems to be described for the first time in the present work.     

Thermal induced mobility of self-assembled platelets of polyethylene-block-poly(ethylene oxide) in liquid precursors of unsaturated polyester thermoset

We report the variation of the molecular assembly and crystallinity of polyethylene-block-poly(ethylene oxide) (EEO, 1400 g mol⁻¹) in a non initiated liquid resin of unsaturated polyester (UP). We particularly focus on the driving force that governs the variation of the molecular assembly of the block copolymer in the UP resin upon heating. For this purpose, we performed a set of experiments combining time resolved in situ SAXS and WAXS measurements upon heating and cooling. Upon heating, SAXS shows that the inter-distance of the EEO domains decreases dramatically for temperature above 75 °C (between 90 and 30 nm for the initial system at room temperature vs 9 nm at 110 °C) suggesting that the initially homogeneously dispersed EEO domains undergo aggregation. This is consistent with the macroscopic phase separation observed in such temperature range. In situ WAXS shows that the onset of the aggregation coincides with the melting of the PE domains suggesting that the flexibility of the platelets plays an important role in their aggregation. Upon cooling, peculiar behaviour is observed for the systems with higher EEO content, with an irreversible structure formation leading to macroscopic EEO rich domains dispersed in continuous UP rich phase.     

Immobilisation of <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> α-amylase and <Emphasis Type="Italic">Aspergillus niger</Emphasis> glucoamylase enzymes as cross-linked enzyme aggregates

Cross-linked enzyme aggregates (CLEA) of Aspergillus oryzea α-amylase (AoAA) and Aspergillus niger glucoamylase (AnGA) were prepared using glutaraldehyde and dextran polyaldehyde as cross-linkers. The maximum activity recoveries for glutaraldehyde cross-linking were 21.8 % and 41.2 %, respectively. The addition of a proteic feeder (bovine serum albumin) exhibited a negative effect on the activity recoveries for both enzymes. Dextran polyaldehyde was used as a cross-linking agent instead of glutaraldehyde to reduce the activity losses. As a result, an activity recovery of 60.0 % was obtained for Aspergillus oryzea α-amylase. On the other hand, no activity recovery was observed for Aspergillus niger glucoamylase due to the latter enzyme’s affinity for dextran.     

High voltage capillary zone electrophoresis: Operating parameters effects on electroendosmotic flows and electrophoretic mobilities

Summary In High Voltage Capillary Zone Electrophoresis a field is applied across a narrow bore capillary filled with electrolyte solution. An electroendosmotic (EEO) flow is generated within this capillary which sweeps solutes along the tube. An absolute method of flow estimation is described, along with some operating parameter effects on the solute mobility. System parameters enabling flow direction reversal and a zero flow are described. The use of several capillaries simultaneously and the effect of pH on EEO flow rates are also shown. Effects of various operating parameters on solute resolution are also detailed.     

Enantioseparation of selected chiral sulfoxides in high‐performance liquid chromatography with polysaccharide‐based chiral selectors in polar organic mobile phases with emphasis on enantiomer elution order

The separation of the enantiomers of 17 chiral sulfoxides was studied on polysaccharide‐based chiral columns in polar organic mobile phases. Enantiomer elution order (EEO) was the primary objective in this study. Two of the six chiral columns, especially those based on amylose tris(3,5‐dimethylphenylcarbamate) and cellulose tris(4‐chloro‐3‐methylphenylcarbamate) (Lux Cellulose‐4) proved to be most successful in the separation of the enantiomers of the studied sulfoxides. Interesting examples of EEO reversal were observed depending on the chiral selector or the composition of the mobile phase. For instance, the R‐(+) enantiomer of lansoprazole eluted before the S‐(?) enantiomer on Lux Cellulose‐1 in both methanol or ethanol as the mobile phase, while the elution order was opposite in the same eluents on amylose tris(3,5‐dimethylphenylcarbamate) with the S‐(?) enantiomer eluting before the R‐(+) enantiomer. The R‐(+) enantiomer of omeprazole eluted first on Lux Amylose‐2 in methanol but it was second when acetonitrile was used as the mobile phase with the same chiral selector. Several other examples of reversal in EEO were observed in this study. An interesting example of the separation of four stereoisomers of phenaminophos sulfoxide containing chiral sulfur and phosphor atoms is also reported here.     

STRUCTURAL CHANGES AND RELEASE CHARACTERISTICS OF CROSSLINKED CHITOSAN BEADS IN RESPONSE TO SOLUTION pH

The present investigation describes a novel method for preparing beads based on crosslinked chitosan with glutaraldehyde interpenetrating glycine polymer network. Four type of beads, viz., CHI1 (composed of chitosan, glycine and glutaraldehyde); CHI2 (composed of chitosan and glutaraldehyde); CHI3 (composed of chitosan and glycine) and CHI4 (only chitosan) were prepared and their release characteristics were studied using thyamine hydrochloride (Thy-HCl) as a model drug. Structural changes during swelling of CHI1 beads in solutions of different pH were studied using IR and UV spectroscopy.     

Optimization of the HPLC enantioseparation of 3,3′‐dibromo‐5,5′‐disubstituted‐4,4′‐bipyridines using immobilized polysaccharide‐based chiral stationary phases

The HPLC enantioseparation of nine atropisomeric 3,3′,5,5′‐tetrasubstituted‐4,4′‐bipyridines was performed in normal and polar organic (PO) phase modes using two immobilized polysaccharide‐based chiral columns, namely, Chiralpak IA and Chiralpak IC. The separation of all racemic analytes, the effect of the chiral selector, and mobile phase (MP) composition on enantioseparation and the enantiomer elution order (EEO) were studied. The beneficial effect of nonstandard solvents, such as tetrahydrofuran (THF), dichloromethane (DCM), and methyl t‐butyl ether on enantioseparation was investigated. All selected 4,4′‐bipyridines were successfully enantioseparated on Chiralpak IA under normal or PO MPs with separation factors from 1.14 to 1.70 and resolutions from 1.3 to 6.5. Two bipyridines were enantioseparated at the multimilligram level on Chiralpak IA. Differently, Chiralpak IC was less versatile toward the considered class of compounds and only five bipyridines out of nine could be efficiently separated. In particular, on these columns, the ternary mixture n‐heptane/THF/DCM (90:5:5) as MP had a positive effect on enantioseparation. An interesting phenomenon of reversal of the EEO depending on the composition of the MP for the 3,3′‐dibromo‐5,5′‐bis‐(E)‐phenylethenyl‐4,4′‐bipyridine along with an exceptional enantioseparation for the 3,3′‐dibromo‐5,5′‐bis‐ferrocenylethynyl‐4,4′‐bipyridine (α = 8.33, R_s = 30.6) were observed on Chiralpak IC.     

Recovery and sporicidal resistance of various B. subtilis spore preparations on porcelain penicylinders compared with results from AOAC test methods

Sporicidal test results obtained from carriers inoculated with 4 types of defined Bacillus subtilis spore preparations were compared with the standard AOAC sporicidal test using soil extract nutrient broth (SENB) B. subtilis 19659 spores. Recoveries of spores inoculated on penicylinders from B. subtilis clean spores (washed and suspended in water) and B. subtilis 19659 spores inoculated from culture filtrates according to the AOAC method were compared. Spores were exposed to 6 concentrations (0.5-3.0% w/v) of glutaraldehyde in phosphate buffer (pH 7.5) for 10 h. Concentrations were established by titrimetry and liquid chromatography. Recoveries of surviving spores were determined for 3 types of clean B. subtilis var. niger preparations, one clean B. subtilis 19659 preparation, and the SENB B. subtilis 19659 filtrates. Spore carriers, inoculated by the standard AOAC protocol, resulted in as much as a 2-log number difference in runs 1-12, but not more than 0.5 log number for each clean spore preparation. The SENB spores varied most in resistance to glutaraldehyde, with no growth in recovery media from 3 different batches of 1, 1.5, and 2% glutaraldehyde. Separate batches of SENB preparations of B. subtilis 19659 were resistant and destroyed by 1.0% glutaraldehyde, with 3.98 and 6.0 log numbers of spores on penicylinders, respectively. Clean spore preparations of B. subtilis 19659 on porcelain penicylinders were more resistant to glutaraldehyde than were SENB spores. Nutrient agar/Mg/Ca and nutrient agar/Mg spore preparations of B. subtilis var. niger showed the most uniform resistance to glutaraldehyde. Spores with calcium added showed increased resistance to glutaraldehyde. B. subtilis 19659 spores from the Columbia broth spore preparation were the most resistant and were recovered after exposure to 3.0% glutaraldehyde.     

Preparation of Cross-Linked Enzyme Aggregates of Trehalose Synthase via Co-aggregation with Polyethyleneimine

Trehalose synthase (TreS) from Meiothermus ruber was co-aggregated with polyethyleneimine (PEI) and precipitated with polyethylene glycol (PEG), followed by cross-linking with glutaraldehyde to obtain TreS-polyethyleneimine cross-linked enzyme aggregates (termed as CLEAs-PEI-PEG). The TreS solution at 0.5 mg mL^?1 protein concentration, with PEI at a mass ratio of 1:0.8 (enzyme/PEI, w/w) and 25 % (w/v) PEG concentration were found to be most adequate for the co-aggregation of TreS. CLEAs-PEI-PEG was most active with glutaraldehyde at a mass ratio of 1:0.5 (enzyme/glutaraldehyde, w/w) to cross-link the co-aggregates. The CLEAs-PEI-PEG prepared in this work had an optimum pH of 6.5 and optimum temperature of 60 °C. For lower concentrations of enzyme, using PEI could enhance the cross-linking efficiency of TreS. The thermal stability and pH tolerance of CLEAs-PEI-PEG were significantly improved. Scanning electron microscopy revealed that the main structure of CLEAs-PEI-PEG showed scaffolding morphology which was constituted by structured ball-like particles with a size of 1–2.5 μm in diameter.     

Zum Reaktionsmechanismus von Glutaraldehyd mit Proteinen

The interaction of glutaraldehyde with model aliphatic amines was studied in order to understand the crosslinking reaction of glutaraldehyde with proteins. The reaction in organic solvents gave N-alkyl-1,4-dihydropyridines and N,N-dialkyl-1,5-diiminopentanes. The isolated products are new or were previously described by us for the first time¹. Hydration of the reaction products led to stable N-alkylpiperidines and N,N-dialkyl-1,5-diaminopentanes. In aqueous solution the reaction depends on thepH: at apH above 7, N-alkyl-1,4-dihydropyridines and at apH below 7, polymers were obtained. For the crosslinking reaction of proteins with glutaraldehyde the following mechanism is proposed: Monomeric glutaraldehyde reacts with the protein to give intermediate N-alkyl-2,6-dihydroxypiperidines. Intramolecular dehydration leads to the corresponding N-alkyl-1,4-dihydropyridines. Condensation of the cyclic monohydrate of glutaraldehyde and N-alkyl-2,6-dihydroxypiperidines gives linear polymeric crosslinks containing -oxo-N-alkylpiperidine units.

Lubig R., Dissertation, RWTH Aachen, 1974.     

Synthesis and characterization of water-swellableα,β-polyasparthydrazide derivatives

, -polyasparthydrazide (PAHy) was crosslinked by glutaraldehyde to form water-swellable materials possessing a three-dimensional molecular network. Different crosslinking degrees were prepared varying glutaraldehyde/PAHy ratio and samples containing 5-fluorouracil were obtained by incorporating the drug into the polymer networks during the crosslinking reaction. All samples were characterized by swelling tests, thermal, x-ray and SEM analysis. Their microstructure was observed through scanning electron microscopy. Furthermore, for samples containing the anticancer drug,in vitro release studies were performed in pH 7.4 buffer solution.     

Oligomerization of Bifunctional Aldehyde in Reactions with Bipolar Ions

Formation of glutaraldehyde oligomers (tri- and pentamers) in reaction of glutaraldehyde with bipolar ions (monoaminoacetic, -aminoglutaric, and ,-diaminocaproic acids and glutatione tripeptide) was studied.     

Direct astatination of a tumour-binding protein, human epidermal growth factor, using nido-carborane as a prosthetic group

We have investigated a method for direct astatine labeling of proteins. Binding sites for astatine were created by coupling of a nido-carborane derivative to a protein, the human epidermal growth factor (hEGF), using two different conjugation methods - by glutaraldehyde cross-linking or by introduction of sulfohydryl groups by Traut's reagent with subsequent linking of ANC-1 with m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester. The conjugates were astatinated using the Chloramine-T method in high yield. The best labeling was obtained by the glutaraldehyde conjugate with an average yield of 68±9%. In vitro stability tests indicated that the glutaraldehyde conjugated label was as stable as hEGF labeled with astatobenzoate.     

Sporicidal testing of commercial germicides using a chemical standard and a calibrated bioindicator

The AOAC sporicidal method uses as a standard the resistance of spores on carriers to 2.5N HCl. This resistance is variable at exposure times ranging from 2 to 20 min. The method described in this paper uses a glutaraldehyde standard and distinguishes various levels of sporicidal activity in the presence of 1-5% glutaraldehyde by using appropriate spore strains, spore preparations, and spore levels. The resistances of 2 Bacillus subtilis 19659 spore preparations cultured in 10% Columbia broth plus manganese and nutrient agar plus minerals, as well as that of B. subtilis var. niger cultured on Lab-Lemco agar, were tested. T-soy broth was a better recovery medium than fluid thioglycollate or modified fluid thioglycollate for B. subtilis 19659 spores exposed to HCl. Sporicidal tests were done on B. subtilis 19659 spores with 2 types of spore preparations. A commercial glutaraldehyde germicide was used for comparison of the sporicidal activity of the glutaraldehyde standard. Two strains of B. subtilis spores and 4 levels of spores (20,000-80,000, 100,000-400,000, 500,000-800,000, and 1,000,000 and up) were removed from check penicylinders from the same batches used for sporicidal tests. B. subtilis var. niger spores were the most resistant to HCl, while B. subtilis 19659 spores were more resistant to glutaraldehyde. Sporicidal activities of a commercial germicide containing 2.5% glutaraldehyde with additives and another containing 5% glutaraldehyde in phosphate buffer were similar. Both totally destroyed high levels of B. subtilis 19659 spores cultured in 10% Columbia broth plus manganese. Results indicate that use of a glutaraldehyde standard, calibrated numbers of spores on penicylinders (bioindicators), and appropriate spore strains and preparations can reduce the variability of sporicidal testing of commercial germicides.     

Effect of linear polymer additives on the electroosmotic characteristics of agarose gels in ultrathin-layer electrophoresis.

Electroosmotic properties of agarose gels with low, medium, high and super high electroendosmosis (EEO) were evaluated based on the apparent electric field mediated mobility of a neutral, fluorescent marker under constant field strength using ultrathin-layer separation configuration. Electroosmotic flow mobility values were measured in different gel concentrations and also in the absence and the presence of various linear polymer additives. Under ultrathin-layer separation conditions, a slight decrease in electroosmotic flow mobility was observed with increasing agarose gel concentration of 1 to 3% for all agarose gels investigated. When linear polymer additives, such as linear polyacrylamide, hydroxyethyl cellulose or polyethylene oxide were added to 1% low electroendosmosis agarose gel, significant reduction of the electroosmotic flow properties were observed with increasing additive concentration. Effect of the intrinsic electroosmotic properties of the various electroendosmosis agaroses on the apparent mobilities and separation performance of double-stranded DNA fragments during automated ultrathin-layer agarose gel electrophoresis was also investigated.     

Enantioseparations of polyhalogenated 4,4’-bipyridines on polysaccharide-based chiral stationary phases and molecular dynamics simulations of selector–selectand interactions

2’-(4-Pyridyl)- and 2’-(4-hydroxyphenyl)-TCIBPs (TCIBP = 3,3’,5,5’-tetrachloro-2-iodo-4,4’-bipyridyl) are chiral compounds that showed interesting inhibition activity against transthyretin fibrillation in vitro. We became interested in their enantioseparation since we noticed that the M-stereoisomer is more effective than the P-enantiomer. Based thereon, we recently reported the enantioseparation of 2’-substituted TCIBP derivatives with amylose-based chiral columns. Following this study, herein we describe the comparative enantioseparation of both 2’-(4-pyridyl)- and 2’-(4-hydroxyphenyl)-TCIBPs on four cellulose phenylcarbamate-based chiral columns aiming to explore the effect of the polymer backbone, as well as the nature and position of substituents on the side groups on the enantioseparability of these compounds. In the frame of this project, the impact of subtle variations of analyte and polysaccharide structures, and mobile phase (MP) polarity on retention and selectivity was evaluated. The effect of temperature on retention and selectivity was also considered, and overall thermodynamic parameters associated with the analyte adsorption onto the CSP surface were derived from van ’t Hoff plots. Interesting cases of enantiomer elution order (EEO) reversal were observed. In particular, the EEO was shown to be dependent on polysaccharide backbone, the elution sequence of the two analytes being P-M and M-P on cellulose and amylose tris(3,5-dimethylphenylcarbamate), respectively. In this regard, a theoretical investigation based on molecular dynamics (MD) simulations was performed by using amylose and cellulose tris(3,5-dimethylphenylcarbamate) nonamers as virtual models of the polysaccharide-based selectors. This exploration at the molecular level shed light on the origin of the enantiodiscrimination processes.     

A new approach to preparation of granulated materials based on chitosan and its imidazole derivative

A new approach to preparation of granulated materials based on chitosan and N-(5-methyl-4-imidazolyl)- methyl chitosan was developed. The procedure is simple and efficient and involves the use of glutaraldehyde as cross-linking agent without using precipitants. The composition and structural features of the materials obtained were determined by elemental analysis and Fourier IR spectroscopy. The dependence of the degree of material swelling on the degree of functionalization with glutaraldehyde was determined. The polymer granules were used as support for Pd0-containing catalysts for reduction of organic compounds.     

Effect of the Presence of Surfactants and Immobilization Conditions on Catalysts’ Properties of <Emphasis Type="Italic">Rhizomucor miehei</Emphasis> Lipase onto Chitosan

Lipase from Rhizomucor miehei (RML) was immobilized onto chitosan support in the presence of some surfactants added at low levels using two different strategies. In the first approach, the enzyme was immobilized in the presence of surfactants on chitosan supports previously functionalized with glutaraldehyde. In the second one, after prior enzyme adsorption on chitosan beads in the presence of surfactants, the complex chitosan beads-enzyme was then cross-linked with glutaraldehyde. The effects of surfactant concentrations on the activities of free and immobilized RML were evaluated. Hexadecyltrimethylammonium bromide (CTAB) promoted an inhibition of enzyme activity while the nonionic surfactant Triton X-100 caused a slight increase in the catalytic activity of the free enzyme and the derivatives produced in both methods of immobilization. The best derivatives were achieved when the lipase was firstly adsorbed on chitosan beads at 4 °C for 1 h, 220 rpm followed by cross-link the complex chitosan beads-enzyme with glutaraldehyde 0.6% v.v^?1 at pH 7. The derivatives obtained under these conditions showed high catalytic activity and excellent thermal stability at 60° and 37 °C. The best derivative was also evaluated in the synthesis of two flavor esters namely methyl and ethyl butyrate. At non-optimized conditions, the maximum conversion yield for methyl butyrate was 89%, and for ethyl butyrate, the esterification yield was 92%. The results for both esterifications were similar to those obtained when the commercial enzyme Lipozyme® and free enzyme were used in the same reaction conditions and higher than the one achieved in the absence of the selected surfactant.     

Sago/PVA blend membranes for the recovery of ethyl acetate from water

Sago starch is a relatively new polymeric material for development of a hydrophilic membrane for dehydration of alcohol/water. In this study sago based membranes were developed through casting technique for the dehydration of ethyl acetate at azeotropic conditions via pervaporation. Sago was blended with polyvinyl alcohol (PVA) to produce blended sago–PVA membranes with improved physical and chemical properties. The membranes were cross-linked using three different approaches; firstly, using glutaraldehyde, secondly using thermal treatment (80 °C) and thirdly by using both glutaraldehyde and thermal treatment. The effects of various cross-linking methods on the intrinsic properties of hydrophilic polymer membrane were investigated. The membranes were characterized using Fourier transform infrared (FTIR), differential scanning calorimeter (DSC) and thermogravimetric analysis (TGA). The effect of operating conditions such as feed temperature and concentration on the separation factor and flux was discussed. Sago starch polymer shows very high performance and very good stability after polymer blending and cross-linking, which is promising for use in industrial applications.