Semi-preparative Separation Research Articles

Cellulose tris(3,5‐dimethylphenylcarbamate)‐based chiral stationary phases as effective tools for enantioselective HPLC separation of structurally different disubstituted binaphthyls

Cellulose tris(3,5-dimethylphenylcarbamate)-based chiral stationary phases (CSPs) were used for a study of the HPLC retention and enantioseparation behavior of 2,2'-disubstituted or 3,2,2'-trisubstituted 1,1'-binaphthyls and 8,3'-disubstituted 1,2'-binaphthyls. The effects of the mobile phase composition in normal- (NP) and reversed-phase (RP) separation modes were investigated. The NP mobile phases contained n-hexane and propane-2-ol at various volume ratios, the RP ones were obtained by mixing acetonitrile with water or a 20 mM phosphate buffer of pH 6.0 or 3.0. The RP separation mode has been found more suitable for enantioresolution of most of the analytes. The best enantioseparation of 2,2'-diacetyl-1,1'-binaphthyl, 2-hydroxy-2'-(phenylamino)-1,1'-binaphthyl-3-carboxylic acid and 2-amino-2'-hydroxy-1,1'-binaphthyl-3-carboxylic acid was obtained in the mobile phase of ACN/20 mM phosphate buffer, pH 3.0, 40/60 (v/v), whereas N-(2'-hydroxy-1,1'-binaphthyl-2-yl)acetamide, N-(3'-methoxy-1,2'-binaphthyl-8-yl)acetamide, and N-(3'-hydroxy-1,2'-binaphthyl-8-yl)acetamide yielded better results in ACN/water at the same v/v ratio. The analyte-CSP interaction mechanism was found to be temperature independent but the enantioresolution improved at an elevated temperature. The mechanism of the enantioselective discrimination is discussed on the basis of the thermodynamic parameters obtained. Semi-preparative separation conditions have been proposed for 2-amino-2'-hydroxy-1,1'-binaphthyl-3-carboxylic acid, N-(3'-methoxy-1,2'-binaphthyl-8-yl)acetamide, and N-(3'-hydroxy-1,2'-binaphthyl-8-yl)acetamide.

HPLC method for enantioselective analysis of cloprostenol

A new HPLC method for the separation and quantification of cloprostenol enantiomers was developed. The optimized separation system consisted of Chiralcel OD-RH column and acetonitrile–sodium dihydrogenphosphate (pH 3.0; 20 mM) (33:67, v/v) as the mobile phase. Baseline resolution of (±)-cloprostenol ( R = 2.16) was achieved and the analysis time did not exceed 10 min. Limits of detection and quantification were units of μmol/l at 274 nm. The respective values decreased an order of magnitude at 210 nm. The R.S.D. values obtained for the retention factor, peak area and peak height of each enantiomer were less than 2%. Conditions for semipreparative separation of the enantiomers can be achieved easily just by a small adaptation of the mobile phase composition.

Enantioselective separation and determination of single nonylphenol isomers

It has recently become clear that an isomer specific view of technical 4-nonylphenol (NP) is absolutely necessary for the evaluation of the biological behavior of NP, raising additional questions concerning enantiomer specific effects. For the first time, in this study enantioselective HPLC was applied to enantiomeric separation of chiral NP isomers. A semipreparative separation of two NP isomers could be achieved. A GC–MS method has been developed for the simultaneous detection of three chiral NP isomers in water samples. Investigation of influent and effluent samples from a wastewater treatment plant in Germany indicated that enantioselective degradation could occur in the environment. In one examined influent, an enantiomer ratio of 1.7 for two different isomers was determined.

Tailored Ring-Opening Metathesis Polymerization Derived Monolithic Media Prepared from Cyclooctene-Based Monomers and Cross-Linkers

The synthesis of ring-opening metathesis polymerization (ROMP) derived monolithic media prepared from cis-cyclooctene (COE) as monomer, tris(cyclooct-4-ene-1-yloxy)methylsilane (CL) as cross-linker, toluene as microporogen, and 2-propanol as macroporogen is described. Monolithic supports with varying composition in terms of COE, CL, microporogen, and macroporogen were prepared using RuCl2(Py)2(1,3-Mes2-imidazolin-2-ylidene)(CHC6H5) (Mes = 2,4,5-trimethylphenyl, Py = pyridine) and pyridine as modulator and characterized via inverse size exclusion chromatography (ISEC) and electron microscopy. Monoliths 3 × 100 mm in size were used for the separation of a mixture of five proteins, i.e., ribonuclease A, lysozyme, insulin, cytochrome c, and myoglobin. Semipreparative separation of these compounds was achieved within less than 150 s by applying gradient elution. Peak half-widths (ω0.5) were <6 s, and resolution (Rs) was >1.2 throughout. Functionalization of the novel support was accomplished in situ and exemplified by grafting various amounts of 7-oxanorborn-5-ene-2,3-dicarboxylic anhydride on the monolithic structure. By this approach, carboxylic acid loadings up to 86 μmol/g, corresponding to 0.71 wt %, were realized.

Synthesis, chiral HPLC resolution and configuration assignment of 1‐phenylglyceryl trinitrate stereomers

As an introductory study of in vitro vasodilating activity, the access to the four stereomers of 1-phenylglyceryl trinitrate is described using achiral and chiral chromatography. For semi-preparative separation of the enantiomers, a Chiralcel OD (250 x 10 mm, 10 microm) was used. Catalytic reduction leading to the corresponding stereomers of 1-phenylglycerol allowed absolute configuration assignments. The same methods were used for the separation and configuration assignment of the enantiomers of 3-phenylpropane-1,2-diyl dinitrate.

A validated higher-performance liquid chromatography method for quantification of cinchonain Ib in bark and phytopharmaceuticals of Trichilia catigua used as Catuaba

The hydroalcoholic extract, prepared from authentic chopped barks of Trichilia catigua, was evaluated by high-performance liquid chromatography using a diode array detector (200–400 mn). The crude extract was purified by rotation locular counter-current chromatography and the chloroform fraction obtained was clean-up by solid-phase extraction. With the aim of getting preliminary structure information on-line, the methanol fraction thus obtained was analyzed by gradient elution using the diode array detector coupled to a mass spectrometer. The presence of flavalignan in this extract was inferred by the chromatographic band, in the total ion current trace, that had an [M–H] − = 451. With this information, cinchonain Ib was isolated as a pure compound from the crude hydroalcoholic extract using a solid-phase extraction procedure for the sample clean-up followed by a semi-preparative separation using the reverse mode of elution. The isolated compound, after complete characterization, was used as an external standard for the development and validation of a method for the analysis of this compound in herbal medicines using the ultraviolet as the detector. The validated method has been successfully applied for quantification of cinchonain Ib in commercialized herbal medicines sold as Catuaba in Brazil and also in standard chopped barks of T. catigua.

Semipreparative Separation and Determination of Eleutheroside E in Acanthopanax giraldii Harms by High-Performance Liquid Chromatography

A method for the isolation, purification, and determination of eleutheroside E in Acanthopanax giraldii Harms, collected in the Sichuan province (China), is established. The water extraction of A. giraldii Harms is pre-isolated using macroporous adsorption resin (D-101) and a C18 solid-phase extraction cartridge, and the enriched extract is purified to give eleutheroside E (syringaresinol-di-O-beta-D-glucoside; liriodendrin) by semipreparative reversed-phase high-performance liquid chromatography. Structure identification is performed by a comparison of IR, 1H-NMR, 13C-NMR, and electrospray ionization-mass spectrometric data with the literature. The final purity of the compound is 97%. Quantitative determination of eleutheroside E in A. giraldii Harms is performed on a Zorbax SB C18 (150- x 4.6-mm i.d., 5 microm) column. The linear range of eleutheroside E is 4.85-194 mg/L (r = 0.9998), and the average recovery is 99.6-101%. The developed method is simple, reproducible, and easy to operate. It is useful for the evaluation of Acanthopanax giraldii Harms.

Separation of atropisomeric 1,4,5,6-tetrahydropyrimidinium salts by chiral HPLC and determination of their enantiomerization barriers

Chiral HPLC separation of a series of novel atropisomeric quaternary ( 1) and ternary ( 2) 1,2-disubstituted 1,4,5,6-tetrahydropyrimidinium salts bearing disymmetric aryl groups in positions 1 and/or 2 is described. A screening of different polysaccharide stationary phases (OD-R, OJ-R and AD-RH) and chromatographic conditions allowed for partial or baseline resolution of 16 over 26 compounds. When a semi-preparative separation was achieved, the corresponding enantiomerization barriers were determined employing the off-column method. The experimental data were compared inter se in order to establish the factors influencing the magnitude of the barriers and with those corresponding to the parent amidines.

Separation and characterization of synthetic impurities of triclabendazole by reversed-phase high-performance liquid chromatography/electrospray ionization mass spectrometry

A simple high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS) method for the separation and characterization of impurities in the synthesis of triclabendazole has been developed. The analytical separation was achieved on a reversed-phase C18 column using acetonitrile and water (60:40, v/v) as mobile solvent at a flow-rate of 1.0 ml/min at 25 °C, and an UV detection at 230 nm. The on-line HPLC/ESI-MS n examinations were performed using ion trap analyzer with extraction ion chromatography (EIC) technique in positive or negative ion modes. The semi-preparative separation was performed with a reversed-phase column using methanol and water (75:25, v/v) as mobile solvent at a flow-rate of 4 ml/min at 25 °C, and an UV detection at 230 nm. Thus, two impurities were detected and identified as 5-chloro-6-(2,3,4-trichlorophenoxy)-2-methylsulfanyl-1 H-benzoimidazole and 5-chloro-6-(2,3-dichlorophenoxy)-1-methyl-2-methylsulfanyl-1 H-benzoimidazole. Meanwhile, some intermediates of impurity-1 in multi-step synthetic reactions, were tracked. Structural elucidation by 1D and 2D NMR and ESI-MS n was discussed.

Preparative—Semipreparative Separations of Peptides

AbstractFor Abstract see ChemInform Abstract in Full Text.

Reduced-bore monolithic silica column modified with C8-TEOS for reversed-phase electrochromatography.

Monolithic silica columns of 2.7 mm ID were prepared and derivatized with C8-TEOS and TEOS by on-column sol-gel reaction. These C8 large diameter monolithic silica columns gave 21 000 theoretical plates for aromatic hydrocarbons in 60% acetonitrile and 40% Tris-HCI buffer. The surface areas as well as the separation reproducibility were improved on coating by the sol-gel approach. Joule heating was greatly reduced by using monolithic columns to which fine quartz sand had been added during column preparation. Since this is a preliminary investigation on a monolithic column with such a large inner diameter, the separation efficiency was not so high as that presently achieved in normal capillary electrochromatography (CEC). However, use of the columns improved sample loadability and concentration detectability of electrochromatography, and semi-preparative separations could be performed.

Preparative resolution of 1-aminoindan-1,5-dicarboxylic acid (AIDA) by chiral ligand-exchange chromatography.

Chiral ligand-exchange chromatography has been shown to be effective in the resolution and semipreparative separation of 1-aminoindan-1,5-dicarboxylic acid (AIDA) enantiomers. In functional activity experiments, only (S)-AIDA was a potent and mGluR1 subtype selective antagonist.

Circular asymmetrical flow field-flow fractionation for the semipreparative separation of particles.

A new technique for the separation and characterization of particles and polymers based on asymmetrical flow field-flow fractionation was developed. The new circular asymmetrical flow field-flow fractionation instrument (CAFFFE) resembles a quasi-parallel arrangement of 12 individual flow channels. As compared to the classical asymmetrical flow field-flow fractionation (AF-FFF), which can be used so far only for analytical separation and characterization of particles and polymers, the CAFFFE allows the introduction of higher amounts of sample into the channel in a single run so that semipreparative to preparative separation becomes possible. This was demonstrated by the separation of polymer latex standards.

Use of semipreparative supercritical fluid chromatography to obtain small quantities of the albendazole sulfoxide enantiomers

The semipreparative separation of the albendazole sulfoxide enantiomers using chiral supercritical fluid chromatography is presented in this work. For this purpose, a modular SFC chromatograph was adapted to work at semipreparative scale and a Chiralpak AD (250×10 mm) column was used. Different injection volumes were evaluated in order to obtain high purities and throughputs. Using the maximum load, it was possible to obtain 37 mg/h of the first eluted enantiomer with a purity of 99.9%, and 36.5 mg/h of the second eluted enantiomer with a purity of 95%.

Analytical and semipreparative separation of the enantiomers of new acetylcholinesterase inhibitors by high-performance liquid chromatography

The resolution of the enantiomers of new acetylcholinesterase inhibitors by high-performance liquid chromatography (HPLC) was investigated on stationary phases containing cellulose tris-(3,5 methylphenylcarbamide) (Chiralcel OD). The effects of the mobile phase on retention, enantioselectivity and resolution were also studied. Ethanol and isopropanol were tested as organic modifiers and the influence of diethylamine was investigated. The effect of temperature on chiral separations was also studieded.

Enantiomeric separation by HPLC of 1,4-dihydropyridines with vancomycin as chiral selector.

The macrocyclic antibiotics represent a relatively new class of chiral selectors in CE, HPLC, and TLC. We have examined the use of the macrocyclic antibiotic vancomycin as a chiral selector in HPLC for the separation of 1,4-dihydropyridines (DHPs) calcium antagonists (CAs). Chromatographic data of six 1,4-dihydropyridine calcium channel blockers obtained on the vancomycin chiral stationary phase (Chirobiotic V) were compared with those obtained on an alpha(1)-acid glycoprotein (AGP) HPLC stationary phase. Optimization of pH and organic modifier was carried out in order to modulate the retention properties of each system. All chiral neutral DHPs were resolved on the AGP column, whereas on Chirobiotic V only basic DHPs showed a split peak. The analytical chromatographic procedure on Chirobiotic V proved suitable for semipreparative separation, since the separation factor on the analytical column was high enough to obtain pure enantiomers with high yields.

Hydrophobic interaction chromatographic separation of proteins in human blood fractions hyphenated to atomic spectrometry as detector of essential elements

The binding of metals to proteins in blood fractions was investigated applying hydrophobic interaction chromatography (HIC) for protein separation and graphite furnace atomic absorption spectrometry (GFAAS) as the element specific detector. For the semi-preparative separation of metalloproteins in erythrocytes and blood plasma, a HIC column (Fractogel EMD Phenyl I (S) 150 mm×10 mm I.D.) was adapted. The separation column was calibrated with the same four standard proteins as used in Pomazal et al. [Analyst 124 (1999) 657]. The sample injection volume and the ammonium sulphate gradient set-up were optimized: 20 or 200 μl, respectively, of blood plasma and of lysed erythrocytes were injected. The separated proteins were collected in 4-ml fractions and analyzed by GFAAS off-line. An optimization of the GFAAS measuring parameters for Cu, Mn, Fe, Zn, Co, Ni, and Cr was performed. For each element, a specific temperature program was optimized with respect to the matrix of the HIC eluate (0.02 M NaH 2PO 4, 1.8 M (NH 4) 2SO 4). The obtained metal profiles of the eluate were compared with the HIC chromatograms. The limits of detection (LOD) for the elements by GFAAS were: 0.5 ng Cu/ml; 0.2 ng Mn/ml; 1 ng Fe/ml; 0.2 ng Zn/ml; 0.12 ng Co/ml; 0.2 ng Ni/ml; 0.16 ng Cr/ml. The GFAAS method enabled the detection of the proteins of interest via the metals.

SEMI-PREPARATIVE HPLC SEPARATION OF TERPENOIDS FROM THE SEED PODS OF HYMENAEA COURBARIL VAR. STILBOCARPA

Column chromatography of the ethyl acetate extract of the seed pod resin of Hymenaea courbaril var. stilbocarpa furnished a fraction containing a mixture of two new clerodanes and a known clerodane diterpene. Due to the similarity of the structures, they could not be separated by ordinary chromatography even using silica gel impregnated with AgNO3. Thus, an intense study was made in order to optimize the semipreparative separation of this mixture by HPLC (high performance liquid chromatograph) using several different stationary and mobile phases. The best conditions for separation of these diterpenes was on octadecyl-bonded silica with methanol:water:formic acid (85:15:1, v/v/v) as mobile phase, flow rate at 2 mL/min, and UV detection at 240 nm. The fractions obtained were analyzed by gas chromatography-mass spectrometry (GC-MS).

High-performance liquid chromatography applications of optical rotation detection with compensation for scattering and absorbance at the laser wavelength

Use of instrumentation developed to enable simultaneous monitoring of optical rotation (OR) and transmittance allows OR measurements to be made in the presence of high levels of absorbance, scattering or other effects that change the intensity of the plane-polarised light at the photodiode detector. This extends the application of OR detection to areas where it was previously difficult. Examples of the application of high-performance liquid chromatography (HPLC) with the improved OR detector include (i) the analytical scale separation of fructose and sucrose and (ii) the semi-preparative separation of enantiomers of warfarin and Trögers base. A signal-to-noise improvement of up to 150% is found when comparing signals with and without correction for transmittance changes. The improved OR detector has been used in series with a UV detector and the system shown to be suitable for on-line measurement of peak purity in separations using a chiral column under overload conditions.

Semi-preparative gas chromatographic separation of all-trans-perhydrotriphenylene enantiomers on a chiral cyclodextrin stationary phase

Enantiomers of all-trans-perhydrotriphenylene (PHTP) were separated by gas chromatography using heptakis(6- O- tert.-butyldimethylsilyl-2,3-di- O-methyl)-β-cyclodextrin (TBDMS-β-CD) as the chiral selector. Conditions for semi-preparative separations were established using a 2 m×2 mm I.D. packed column and subsequently extended to a 1.8 m×4 mm I.D. column which enabled separations on a mg scale. The column packing was TBDMS-β-CD dissolved in SE-54 coated on Chromosorb P AW-DMCS 80–100 mesh. Optimization of the chromatographic conditions (oven temperature, carrier gas flow, and column load) with respect to better efficiency and peak retention resulted in a system capable of separating up to 10 mg of the racemate per day. Purities of separated enantiomers were determined by capillary gas chromatography. Yields and purities of the fractions obtained by single- and double-step separations are compared. Highly enriched enantiomers with purities of up to 99.6% (99.2% ee) were obtained by a single separation step.