High-performance liquid chromatographic enantioseparations on capillary columns containing monolithic silica modified with cellulose tris(3,5-dimethylphenylcarbamate)

Monolithic capillary columns containing native silica gel were modified with cellulose tris(3,5-dimethylphenylcarbamate) (CDMPC) and used for enantioseparations in capillary liquid chromatography. The method adopted for in situ enantioselective modification of monolithic fused silica capillary columns by coating with CDPMC appears to be fairly simple and fast. High efficiency enantioseparations of test racemic compounds and s(everal chiral drugs were achieved in a short time. It was possible to increase the amount of chiral selector present by multiple coating of monoliths with CDMPC. The baseline enantioseparation of 2,2,2-trifluoro-1-(9-anthryl)ethanol was achieved in an analysis time less than 30 s with this capillary column. In addition, reproducible enantioseparations were obtained when the chiral selector was removed from the monolithic column by flushing it with appropriate solvent and the column recoated.     

High-performance liquid chromatographic enantioseparations on capillary columns containing crosslinked polysaccharide phenylcarbamate derivatives attached to monolithic silica

Monolithic capillary columns containing native silica gel were covalently modified with 3,5-disubstituted phenylcarbamate derivatives of cellulose and amylose and applied for enantioseparations in capillary LC. The method previously used for covalent immobilization of polysaccharide phenylcarbamate derivatives onto the surface of microparticulate silica gel was successfully adapted for in situ modification of monolithic fused-silica capillary columns. The effects of the nature of polysaccharide and the substituents, as well as of multiple covalent immobilization of polysaccharide derivative on chromatographic performance of capillary columns were studied. The capillary columns obtained using this technique are stable in all solvents commonly used in LC and exhibit promising enantiomer resolving ability.     

High-performance liquid chromatographic evaluation of a coated cellulose tris(3,5-dimethylphenylcarbamate) chiral stationary phase having a small-pore silica support

A two-step coating/precipitation synthetic procedure has been developed for the preparation of cellulose tris(3,5-dimethylphenylcarbamate) chiral stationary phase (CSP) having a small-pore silica support. With this synthetic strategy, monodisperse, spherical CSP particles can be produced without the need for a wasteful and time-consuming sieving process. The performance of the synthesized CSP towards a variety of racemates was evaluated in the normal-phase HPLC mode. HPLC separation experiments revealed that the synthesized CSP exhibited a chiral recognition ability fully comparable to the corresponding commercial columns prepared using conventional large-pore silica as the support. Moreover, the synthesized CSP was successfully applied to semipreparative enantioseparation of a new triazole antifungal agent.     

Fast enantioseparations of basic analytes by high-performance liquid chromatography using cellulose tris(3,5-dimethylphenylcarbamate)-coated zirconia stationary phases

In this work, we study the influence of the mobile phase and column temperature on the enantioresolution of basic compounds on microparticulate porous zirconia coated with cellulose tris(3,5-dimethylphenylcarbamate) (CDMPC). The chiral analytes are amino compounds, including a number of beta-blockers. Analytes are eluted with hexane-alcohol mobile phases. We investigated the effect of alcohol (type and concentration), basic eluent additives, and column temperature on the parameters that control resolution (column efficiency, retention and selectivity). Conditions for achieving an adequate separation in the least time have been determined for numerous racemic mixtures. For most solutes, baseline resolution of the enantiomeric pair was achieved in less than 1 min; 12 of 13 pairs were separated in less than 2 min.     

Amylose-3,5-dimethylphenylcarbamate immobilized on monolithic silica stationary phases for chiral separations in capillary electrochromatography

Chiral monolithic silica capillary columns were prepared by immobilization of amylose-3,5-dimethylphenylcarbamate (ADMPC) bearing a small fraction of 3-(triethoxysilyl)propyl residues through intermolecular polycondensation of the triethoxysilyl groups. The obtained columns were used for chiral separations in capillary electrochromatography (CEC). The effects of the silica monolith nature and the used chiral selector concentration on the resulting enantiomeric separations were investigated. Fifteen chiral compounds, including acidic, neutral, and basic substances were evaluated and twelve showed partial or baseline separation at some of the different conditions tested. These results demonstrated the promising applicability of ADMPC-immobilized monolithic silica columns in CEC enantioseparations, but also revealed the need for further improvements on the level of baseline separations and efficiencies.     

High-performance liquid chromatographic separation of carbohydrates on graphitized carbon columns

Graphitized carbon columns (GCC) for high-performance liquid chromatography are relatively new and have a unique ability to resolve isomeric and closely related compounds. The retention mechanism of carbohydrates on GCC is mainly based on adsorption and the flat surface of GCC packing brings about unique selectivity, but also includes hydrophobic interactions. The chromatographic behaviour of monosaccharides, disaccharides, cyclodextrins (CDs), branched CDs, oligosaccharide alditols, chito-oligosaccharides, N-linked oligosaccharides and glycopeptides has been studied, and it has become apparent that the elution patterns are based on the size and planarity of the molecule (position and configuration of linkage).     

Preparation of monolithic silica columns for high-performance liquid chromatography

Preparation methods of monolithic silica columns for HPLC including the surface modification were reviewed. Chemical modification methods recently reported to obtain stationary phases for reversed-phase (RP), chiral, ion-exchange, and hydrophilic interaction chromatography (HILIC) separations were discussed. Recent results related to preparation methods of monolithic silica were also covered. The characteristics and properties of silica monoliths and some applications of monolithic silica columns for different analytical and bioanalytical fields will be commented.     

Preparation and chiral recognition ability of cellulose 3,5-dimethylphenylcarbamate immobilized on silica gel through radical polymerization

Polysaccharide-immobilized chiral stationary phases (CSPs) were prepared by the polymerization of cellulose 3,5-dimethylphenylcarbamate, having a polymerizable vinyl group, such as 4-vinylphenylcarbamate or 2-methacryloyloxyethylcarbamate, at the 6-position, with a vinyl monomer, such as styrene, isoprene, t-butyl acrylate, or t-butyl methacrylate, on silica gel under various conditions. Their chiral recognition abilities were then evaluated with high-performance liquid chromatography. The immobilized cellulose 3,5-dimethylphenylcarbamate remained on the silica gel even if washed with tetrahydrofuran, which could dissolve the cellulose derivative. The chiral recognition abilities of the immobilized CSPs were similar to those of the coated CSPs when the vinyl monomer content was low. The chiral recognition abilities of the obtained immobilized CSPs slightly depended on the vinyl monomers. The immobilization of the cellulose derivatives was more efficiently attained on the silica gel modified with a vinyl compound. The cellulose derivatives, randomly having a vinyl group at the 2-, 3-, or 6-position of the glucose unit, were prepared by a one-pot reaction. The immobilization efficiency of these derivatives was slightly lower than that of the derivative with the vinyl group at the 6-position. © 2003 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 41: 3703–3712, 2003     

Efficiency of methacrylate monolithic columns in reversed-phase liquid chromatographic separations

Butyl-methacrylate-based porous monoliths were prepared inside fused-silica capillaries as reversed-phase separation media for liquid chromatography (LC) and capillary electrochromatography (CEC). During our previous research on methacrylate-based monoliths for reversed-phase separations, we noticed that a separation efficiency of up to 300,000 plates/m can easily be obtained in the CEC mode for unretained compounds. However, the efficiencies for retained compounds were much lower in reversed-phase systems, especially in pressure-driven LC. In this work methacrylate-based columns were prepared and characterized in terms of efficiency and retention in reversed-phase (pressure-driven) LC and in CEC. Much attention has been paid to the mass-transfer mechanism in the stationary phase. Factors that affect the plate heights for specific compounds have been investigated. A possible explanation for the relatively low separation efficiency of retained compounds and suggestions to improve molecular mass transfer are provided.     

High-performance liquid chromatographic resolution of enantiomers on chiral amine-bonded silica gel

Summary A chiral stationary phase with an immobilized, optically active diamine was prepared for the separation of enantiomers. The synthesis of the phase was carried out by bonding (–)trans-1,2-cyclohexanediamine to microparticulate silica gel through the coupling agent 3-glycidoxypropylsilane. The resolution of the racemic compounds catechin, 2,2-dihydroxy-1,1binaphthyl and trans-1,2-cyclohexandiol, is reported.     

Enantioselectivity of monolithic silica stationary phases immobilized with different concentrations cellulose tris (3,5-dimethylphenylcarbamate), analyzed with different mobile phases in capillary electrochromatography

The 3,5-dimethylphenylcarbamate derivatives of cellulose bearing 3-(triethoxysilyl)propyl residues were immobilized in a capillary format onto a monolithic silica support by intermolecular polycondensation of the triethoxysilyl groups. The resulting columns were used for chiral separations using capillary electrochromatography. The effects of the synthesizing solvent, the selector coating procedure, the chiral selector concentration onto the silica monolith and the mobile phase pH value, on the separation of enantiomers were studied. The column-to-column reproducibility and stability also were evaluated. A test set of 14 chiral substances, including acidic, neutral, bifunctional and basic compounds, was used to investigate the effects of the factors mentioned above. Twelve pairs of enantiomers showed enantioselectivity at some of the different conditions tested. The column-to-column repeatability was satisfactory, and the prepared columns were stable under the adopted analysis conditions.     

Influence of vinyl monomers and temperature on immobilization of cellulose 3,5-dimethylphenylcarbamate onto silica gel as chiral stationary phases for high-performance liquid chromatography

Cellulose 3,5-dimethylphenylcarbamates bearing a low content of a vinyl group at the 6-position on the glucose units were synthesized by a previously developed regioselective method and chemically immobilized onto a vinylized silica gel as chiral stationary phases (CSPs) for high-performance liquid chromatography (HPLC). The immobilization of the derivatives was performed through a radical polymerization reaction with AIBN as the initiator in the presence of toluene. The effects of vinyl monomers, such as isoprene, 2,3-dimethyl-1,3-butadiene (DMBD), ethylene glycol dimethacrylate (EDMA) and 1,5-hexadiene, on the immobilization and enantioselectivities of the derivatives were investigated. The effect of the temperature used for the radical polymerization reaction on the immobilization was also examined. In addition, the direct comparison of the chiral recognition abilities of the laboratory-made and commercially available columns was discussed.     

Enantiomer separation of dimethyl dicarboxy alpha-biphenyl (DDB) and its analogues on a covalently bonded cellulose tris-(3,5-dimethylphenylcarbamate) CSP

Dimethyl dicarboxy alpha-biphenyl (DDB) and its analogues represent atropisomers which have been resolved on the covalently bonded cellulose tris-(3,5-dimethylphenylcarbamate) (CDMPC) CSP. Different kinds of alcohols, tetrahydrofuran (THF), and chloroform were employed as mobile phase modifiers (MPMs), and their influence on the retention and separation of the enantiomers was investigated. Ternary mobile phases (hexane/2-propanol/THF, hexane/2-propanol/chloroform) were employed to investigate the separation of the five enantiomers. The advantages of the broader choice of solvents offered by the covalently bonded CDMPC CSP were discussed. The effect of structural variation of the enantiomers on their retention and separation was investigated.     

Monolithic silica capillary column with coated cellulose tris(3,5-dimethylphenylcarbamate) for capillary electrochromatographic separation of enantiomers

Monolithic silica capillary columns were prepared by a sol-gel process in fused-silica capillaries with an inner diameter of 50 microm and were modified by coating of cellulose tris(3,5-dimethylphenylcarbamate). Influences of the factors in the modification process on enantiomer separations were investigated. The prepared columns were used to perform enantiomer separations by CEC. Fifteen and two pairs of enantiomers were separated under aqueous and nonaqueous mobile phases, respectively, and most of them were baseline-separated with very high column efficiencies. The Van Deemter curve was found flat under high linear velocity of the mobile phase, which indicated favorable kinetic properties of the prepared columns. Baseline separation of a pair of enantiomers was achieved in 90 s with high-column efficiency by short-end separation under high voltage.     

High-performance liquid chromatographic resolution of reverse isomers of 1,2-diacyl-rac-glycerols as 3,5-dinitrophenylurethanes

The resolution of reverse isomers remains a major unsolved problem in glycerolipid chromatography. We have investigated the separation of the reverse isomers of 1,2-diacyl-rac-glycerols under a variety of high-performance liquid chromatography (HPLC) conditions. The reverse isomers of diacylglycerols having various pairs of acyl groups including short and highly unsaturated chains, which were prepared by partial Grignard degradation of the corresponding triacylglycerols, were chromatographed as 3,5-dinitrophenylurethanes. Excellent resolution was achieved for the reverse isomers of very different pairs of acyl groups, such as acetate-palmitate and docosahexaenoate-palmitate, by chiral-phase HPLC on columns containing (R)- and (S)-1-(1-naphthyl)ethylamine polymeric phases, reversed-phase HPLC on a highly efficient C18 column (4 microm particle size) and silver ion HPLC on a silver loaded cation-exchange column. The chiral-phase HPLC also permitted complete enantiomer resolution for all the reverse isomers examined. No satisfactory resolution by any of the HPLC methods, however, was obtained for the reverse isomers possessing minor differences in chain lengths and degree of unsaturation, such as laurate-palmitate and oleate-linoleate. The limitations of resolution and characteristics of elution are described.     

Photopolymerized monolithic capillary columns for rapid micro high-performance liquid chromatographic separation of proteins

The preparation of monolithic poly(butyl methacrylate-co-ethylene dimethacrylate) capillary columns using photoinitiated in situ polymerization within 200 microm i.d. capillaries and their application for microHPLC separations of proteins have been studied. The low resistance to flow characteristic of monolithic columns, enabled the use of very high flow rates of up to 100 microL/min representing a flow velocity of 87 mm/s. Very good separations of a model protein mixture consisting of ribonuclease A, cytochrome c, myoglobin, and ovalbumin was achieved in less than 40 s using a very simple single step gradient of the mobile phase. Interestingly, no effect of the pore size on the separations of proteins was observed for these monolithic columns within the size range of 0.66-2.2 microm. The monolithic microHPLC columns are found very robust and no changes in the long term separation performance and back pressure were observed.     

High-performance liquid chromatographic separation of some 2- and 4-hydroxy derivatives of 2'-benzoylbenzoic acid on Florisil and silica gel columns

A series of 2'-benzoylbenzoic acid derivatives including some pairs of positional isomers and other related compounds were chromatographed by HPLC using a normal-phase system with polar adsorbents (Florisil and silica) and 2-propanol, 1,4-dioxane and tetrahydrofuran as modifiers of the eluent. The results obtained were compared as log k′_Florisil vs. log k′_silica relationships to ascertain the differences in distribution and chemical character of surface-active centres of the investifated adsorbents and the mechanism of elution in different eluent systems     

Sub-1-micron mesoporous silica particles functionalized with cyclodextrin derivative for rapid enantioseparations on ultra-high pressure liquid chromatography

Mesoporous silica particles of relatively uniform sub-1-micron size (0.6-0.9 μm) were successfully prepared by a modified synthesis strategy and applied in chiral separation in an ultra-high pressure liquid chromatography system. These particles were prepared via a ternary surfactant system (Pluronic P123, F127 and hexadecyltrimethyl-ammonium bromide) and subsequently derivatized with perphenylcarbamoylated-β-cyclodextrin moieties. The mesoporous silica particles, despite their submicron size, enabled low back-pressure operation on an ultra-high pressure liquid chromatography system at a maximum flow rate of 2 ml/min. In addition, the particles possessed high surface area (480 m(2)/g) and thus afforded high cyclodextrin derivative loading (32 μmol/g), demonstrating rapid enantioseparation and good resolution of 6 basic and neutral racemates.