Separation of geometrical isomers of sesquiterpene and diterpene alcohols on bare silica by carbon dioxide supercritical fluid chromatography

Summary The application of supercritical fluid chromatography (SFC) with modified carbon dioxide to the separation of geometrical isomers of several sesquiterpene or diterpene alcohols is reported. The chromatographic system consists of bare silica (250×4.6 mm i. d.) with carbon dioxide-methanol mobile phase and UV detection. A mixture of sesquiterpene alcohols, (2E-6E)-farnesol, -bisabolol, (E)-nerolidol and (Z)-nerolidol, and also of two diterpene alcohols, (R)- and (S)-geranyllinalol, was separated by SFC using a mixture of carbon dioxide modified with 0.5–1.0% methanol as mobile phase.     

Supercritical fluid chromatography of metoprolol and analogues on aminopropyl and ethylpyridine silica without any additives

Metoprolol and a number of related amino alcohols and similar analytes have been chromatographed on aminopropyl (APS) and ethylpyridine (EPS) silica columns. The mobile phase was carbon dioxide with methanol as modifier and no amine additive was present. Optimal isocratic conditions for the selectivity were evaluated based on experiments using design of experiments. A central composite circumscribed model for each column was used. Factors were column temperature, back-pressure and % (v/v) of modifier. The responses were retention and selectivity versus metoprolol. The % of modifier mainly controlled the retention on both columns but pressure and temperature could also be important for optimizing the selectivity between the amino alcohols. The compounds could be divided into four and five groups on both columns, with respect to the selectivity. Furthermore, on the aminopropyl silica the analytes were more spread out whereas on the ethylpyridine silica, due to its aromaticity, retention and selectivity were closer. For optimal conditions the column temperature and back-pressure should be high and the modifier concentration low. A comparison of the selectivity using optimized conditions show a few switches of retention order between the two columns. On aminopropyl silica an aldehyde failed to be eluted owing to Schiff-base formation. Peak symmetry and column efficiency were briefly studied for some structurally close analogues. This revealed some activity from the columns that affected analytes that had less protected amino groups, a methyl group instead of isopropyl. The tailing was more marked with the ethylpyridine column even with the more bulky alkyl substituents. Plate number N was a better measure than the asymmetry factor since some analyte peaks broadened without serious deterioration of symmetry compared to homologues.     

Assay of tolnaftate and related impurities by isocratic supercritical fluid chromatography

Tolnaftate, an antifungal drug (TF) and related impurities arising from synthesis, viz., N-methyl-m-toluidine (NMmT) and beta-naphthol-1-chlorothio carbamate (beta-NCTC) can be determined by supercritical fluid chromatography. Even though it was possible to elute TF completely with neat SCF CO2, the peaks of the impurities were found to merge. The chromatographic figures of merit of the three analytes such as retention time (tR), capacity factor (k), selectivity factor (alpha), no. of theoretical plates (N), were optimized. The three compounds can be resolved in 5 min on a Hypersil (250 x 4.0 mm) 5 mu, C18 column with supercritical carbon dioxide, modified with 1.96% methanol as the mobile phase at 9.81 MPa and at 40 degrees C. Detection was carried out at 220 nm. The data as evaluated by the linear regression least squares fit method gave linearity ranges from 0.2 to 10.0 microg/mL for TF and NMmT and 0.3 to 10.0 microg/mL for beta-NCTC with correlation coefficients > 0.99. The method was successfully employed to estimate levels of 0.01% for NMmT and 0.02% for beta-NCTC with respect to TF.     

Assay of tolnaftate and related impurities by isocratic supercritical fluid chromatography

Tolnaftate, an antifungal drug (TF) and related impurities arising from synthesis, viz., N-methyl-m-toluidine (NMmT) and β-naphthol-1-chlorothio carbamate (β-NCTC) can be determined by supercritical fluid chromatography. Even though it was possible to elute TF completely with neat SCF CO₂, the peaks of the impurities were found to merge. The chromatographic figures of merit of the three analytes such as retention time (t_R), capacity factor (k^|), selectivity factor (α), no. of theoretical plates (N), were optimized. The three compounds can be resolved in 5 min on a Hypersil (250 × 4.0 mm) 5 μ, C₁₈ column with supercritical carbon dioxide, modified with 1.96% methanol as the mobile phase at 9.81 MPa and at 40?°C. Detection was carried out at 220 nm. The data as evaluated by the linear regression least squares fit method gave linearity ranges from 0.2 to 10.0 μg/mL for TF and NMmT and 0.3 to 10.0 μg/mL for β-NCTC with correlation coefficients > 0.99. The method was successfully employed to estimate levels of 0.01% for NMmT and 0.02% for β-NCTC with respect to TF.     

Direct sample injection in supercritical fluid chromatography with packed fused silica column

A direct sample injection technique was developed for supercritical fluid chromatography in a packed capillary column, with carbon dioxide as mobile phase and a flame ionization detector. The method allowed solutions, neat liquids, and even solids to be introduced as samples. Also, extraction with supercritical carbon dioxide was combined with this method to separate polymer additives.     

Chiral separation of omeprazole and several related benzimidazoles using supercritical fluid chromatography

A study of the enantiomeric separation of omeprazole and several related benzimidazoles, using supercritical fluid chromatography (SFC), on the amylose based column Chiralpak AD is presented in this work. The effect of the organic modifier as well as temperature on the retention and enantioresolution was investigated. Alcohol-type modifiers provided the best results, allowing the enantiomeric separation of all the compounds studied with resolutions that were in most cases higher than 2, and analysis times lower than 10 minutes. An investigation of the temperature effect revealed that the isoelution temperature was below the working temperature range in only two cases, and hence it was better to work at the highest temperature permitted.     

Polymethylhydrosiloxane surface deactivation of silica particles for packed capillary column supercritical fluid chromatography

Summary Spherical porous silica particles (10 μm diameter, 300 ? and 80 ? pores), spherical nonporous silica particles (10 μm diameter), and irregular porous silica particles (≈ 10 μm diameter, 80 ? pores) were deactivated with polymethylhydrosiloxane (PS). The surface activities of the deactivated silica particles were investigated using various polar compounds under supercritical fluid chromatography (SFC) conditions (neat CO₂), and compared with a commercial C₁₈-bonded phase. The small pore (80 ?) silica particles could be more completely deactivated than larger pore (300 ?) and nonporous silica particles. The success of the PS deactivation method is ascribed to the excellent match between the reactive groups on the polymer backbone and the silica surface, and the formation of a highly crosslinked polymeric layer over the surface. Physical processes, such as adsorption and desorption of the deactivation reagent on the surface and diffusion from the surface, were found to have important effects on the deactivation. Using capillary columns packed with PS deactivated silica particles, typical polar organic compounds, including hydroxyl-containing compounds, carbonyl-containing compounds, free amines, and free carboxylic acids, were separated by SFC and compared with results from a commercial C₁₈-bonded phase. While the results clearly show that the PS deactivated particles were more inert than the C₁₈-bonded phase, better deactivation methods are still needed for separation of free acids and alkylamines.     

Analysis of sulphonamides using supercritical fluid chromatography and supercritical fluid chromatography-mass spectrometry

Packed-column supercritical fluid chromatography has been used for the separation of mixtures of sulphonamides on silica and amino-bonded stationary phases utilizing carbon dioxide with methanol modifier as the mobile phase. The effect of modifier concentration, column pressure and modifier identity on retention was also studied. Packed-column supercritical fluid chromatography-mass spectrometry (SFC-MS) of these mixtures utilizing both moving-belt and modified thermospray interfaces was also studied. The identification of sulphamethazine in a spiked porcine kidney extract was performed by SFC-MS using the moving-belt interface.     

End-functionalized polyethylene oxide coated silica particles for packed capillary column supercritical fluid chromatography

Summary Polyethylene oxide (PEO)-based polymers with hydroxy, methoxy, and aminopropoxy terminal groups were coated on diol functionalized and hexamethyldisilazane end-capped silica particles. Proton-donor and proton-acceptor test solutes, including carboxylic acids, hydroxy-containing compounds, arylamines, and alkylamines were used to evaluate the chromatographic performances of these polymer coated particles under SFC conditions with neat CO₂ as mobile phase. It was found that the particles coated with hydroxy-terminated PEO were suitable for the separation of proton-donor compounds such as hydroxy-containing compounds and carboxylic acids, and the particles coated with aminopropoxy-terminated PEO could be used for the separation of amines. That is, the proton-accepting stationary phase is suitable for the separation of proton accepting solutes, including strong basic alkylamines (pK_b4), using neat CO₂ as mobile phase, while the protondonating stationary phase is suitable for the separation of proton-donating compounds such as carboxylic acids (pK_a4). Hydrogen bond basicity was found to be a critical factor for the chromatography of basic amines. Low volatility acidic and basic drugs were chromatographed using the new stationary phases. The stability of the PEO coated particles was determined by measuring the loss of organic carbon under SFC conditions. It was found that approximately 18 % of the coating (average molecular weight of 15,000) was washed out of the particles by supercritical CO₂ after 7 h at 350 atm and 50°C     

Separation of salbutamol and six related impurities by packed column supercritical fluid chromatography

Summary A rapid separation of salbutamol sulphate and six related impurities: 5-formyl-saligenin, salbutamol ketone, salbutamol bis ether, isopropyl salbutamol, desoxysalbutamol sulphate and salbutamol aldehyde, has been achieved by employing packed column supercritical fluid chromatography. The effects of temperature, pressure, additive concentration and identity on retention have been studied. The use of a basic additive is necessary in order to elute the compounds and improve the peak shape. The best results were obtained by using a diol column and a gradient of modifier (methanol with 0.5% of n-propylamine).     

Purification of OV-17 by supercritical fluid fractionation for fused silica capillary gas chromatography

Commercially available OV-17 was partitioned into a low and a higher molecular weight fraction by supercritical fluid extraction with carbon dioxide. This higher molecular weight fraction is more thermally stable than the original OV-17 due to its more viscous and gum-like nature. Based on the growing evidence in the literature that a deactivating agent for fused silica open tubular columns should be compatible with the stationary phase, a precoat of 1,3,5-trimethyl-1,3,5-triphenylcyclotrisiloxane was applied prior to the static coating of high molecular weight OV-17. Fused silica OV-17 columns prepared in this fashion are stable up to 300°C.     

Capillary supercritical fluid chromatography of limonoids

Capillary supercritical fluid chromatography (SFC) with carbon dioxide as the mobile phase has been used to separate a mixture of limonoid standards. Chromatographic conditions were optimized to enable analysis of plant extracts. The seed of Aphanamixis polystacha (Meliaceae) and the stem bark of Harpephyllum caffrum (Anacardiaceae) were extracted with hexane and the extracts were analyzed by SFC to demonstrate the potential of the technique as a screening tool. SFC analysis indicated the presence of limonoids in the hexane extract of the bark of Entandrophragma delevoyii (Meliaceae). One limonoid and two protolimonoids isolated from the extract were separated by SFC.     

Calculation of amino acid hydrophilicity indices for retention of peptides on amide, diol and silica columns in normal-phase liquid chromatography

Summary A set of hydrophilicity parameters in a normal-phase liquid chromatography of peptides is presented in order to clarify the contribution of individual amino acid residues to peptide retention and to predict retention times. The retention of 100 peptides was studied using normal-phase liquid chromatography on amide, diol and silica columns. An acetonitrile-water mixed solution containing 0.2% trifluoroacetic acid +0.2% triethylamine was used as the mobile phase in a linear gradient elution system. The contribution of each residue upon retention was calculated by linear multiple regression analysis. This paper described the contribution values as “hydrophilicity retention coefficients”. Using these hydrophilicity retention coefficients, retention times could be predicted for peptides of known amino acid content and sequence. A set of hydrophilicity retention coefficients on each column was successfully explained by contributions to the degree of retention.     

Apparatus and separations in supercritical fluid chromatography

An apparatus for the chromatography with supercritical dense gases (supercritical fluids) has been designed, the characteristic feature being a pressure cascade. Three decreasing pressure levels containing the supply part, the chromatographic column, and the detector, respectively are controlled by feed back loops. Independent control and programming of pressure, temperature, and feed rate thereby become possible. Separations of oligostyrenes with n-pentane/methanol as the mobile phase and porous silica as the stationary phase were carried out on analytical and preparative scales. The resolution appears to be superior to that obtainable with liquid chromatography. Mass spectra of the peaks showed that oligomeric species of 1 monomer weight are cleanly separated. The influence of the parameters, particularly pressure and temperature, on the efficiency of the separation was studied. Slow upward pressure and temperature programming were found beneficial for the separation.