Paper electrophoresis with surface-enhanced fluorimetric detection

Amorphous fumed silica was used as a fluorescence sensitizer coated on a filter-paper substrate for use in paper electrophoresis. The usefulness of the separation method was illustrated for a mixture of two polynuclear aromatic compounds of biological interest: benzo[a]pyrene (BaP) and benzo[a]pyrene-r-7,t-8,9,10-tetrahydrotetrol, a hydrolysis product of BaP—DNA adduct. These two compounds were well separated and an enhancement of the fluorescence intensity was also observed when the sensitizer was applied to filter-paper prior to electrophoretic separation.     

Assay of N-Propylajmaline in Blood Plasma by Ion-Pair Liquid-Liquid Chromatography

Abstract

A sensitive method for assay of N-propylajmaline (prajmaline) in human plasma is described. The quaternary ammonium compound exists as a pair of stereoisomers, which are isolated and separated by ion-pair liquid-liquid chromatography on microporous silica particles. An aqueous solution containing perchloric acid and sodium perchlorate is used as stationary phase and a mixture of butanol, dichloroethane and hexane as mobile phase. The procedure involves ion-pair extraction from plasma and evaporation prior to the chromatographic separation. Selective detection is achieved by using a fluorescence detector. The method allows assay of concentrations down to 10 pmol of the two forms of prajmaline in 1 ml of plasma with a relative standard deviation below 5 %.     

Determination of benzo[a]pyrene in cigarette smoke condensate by liquid chromatography on amberlite xad-2

A method is described for the determination of benzo[a]pyrene in cigarette smoke condensate which utilizes chromatographic fractionation on Amberlite XAD-2. PAH are initially separated by step-wise gradient elution, progressing from reverse to normal-phase modes of operation. Other separation steps involve automated column chromatography on silica gel and thin-layer chromatography on 20% acetylated cellulose. Benzo[a]pyrene is finally determined by u.v. spectrophotometry and liquid scintillation counting of ¹⁴C—benzo[a]pyrene tracer. Results obtained compare favorably with those of the more traditional liquid—liquid extraction methods.     

Sensitive and Selective Fluorescence Detection of Phenoxyacid Herbicides by Liquid Chromatography with On-Line Ion-Pair Extraction

Abstract

Fluorescent probes, especially a newly synthesized N-substituted 1-cyanobenz[f]-isoindole quaternary ammonium fluorophore, were used as counter ions in a reaction detector for on-line ion-pair extraction of phenoxyacid herbicides. The probe was used in an on-line post-column set-up coupled to a reversed-phase chromatographic system. After separation on an C-8-bonded silica column using an aqueous methanol (pH 2.5) mobile phase, the herbicides were on-line deprotonated by post-column addition of a 10mM sodium phosphate buffer (pH 8.0), in which the probe was dissolved. Subsequently, the ion-pairs were extracted on-line with chloroform-1-butanol (80:20, v/v) and were monitored by fluorescence detection. Using this system, at least seven herbicides could be separated. The detection limits of 2,4-dichlorophe xyacetic acid and 2,4,5-trichlorophenoxyacetic acid were 400 pg (S/N = 3). The repeatability, based on peak height measurements, for 100ng injections was about 0.5%. Calibration curves were linear over the investigated range of 1–100ng, with correlation coefficients of 0.999 for the two analytes. Application to a drinking water sample is presented.     

Instrumental planar chromatographic method for determination of carbamazepine in human serum

An instrumental planar chromatographic (HPTLC) method for quantification of carbamazepine in human serum was developed using liquid‐liquid extraction with dichloromethane, fluorescence activation with perchloric acid 60%/ethanol/water (1:1:1, v/v) and fluorescence detection. Planar chromatographic separation was performed on precoated silica gel F254 HPTLC plates using a mixture of ethyl acetate/toluene/methanol/acetic acid glacial (5:4:0.5:0.5, v/v) as mobile phase. Densitometric detection was done at 366 nm. The method was validated for linearity, precision and accuracy. Linear calibration curves in the range of 3 and 20 ng/μL showed correlation coefficient of 0.998. The intra‐assay and inter‐assay precision, expressed as the RSD, were in the range of 0.41–1.24% (n = 3) and 2.17–3.17% (n = 9), respectively. The LOD was 0.19 ng, and the LOQ was 0.57 ng. Accuracy, calculated as percentage recovery, was between 98.98 and 101.96%, with a RSD not higher than 1.52%. The method was selective for the active principle tested. In conclusion, the method is useful for quantitative determination of carbamazepine in human serum.     

Preparation and Evaluation of P-tert-Butyl-calix[8]arene-bonded Silica Stationary Phase for High Performance Liquid Chromatography

ABSTRACT

A new p-fert-butyl-calix[8]arene-bonded silica gel stationary phase was synthesized through heterogeneous functionalisation of suspended porous silica. A characterization of its structure was carried out by using elemental analysis, FTIR and ¹³C solid state NMR spectroscopy. Chromatographic performance of the new packing material was investigated by employing polycyclic aromatic hydrocarbons (PAHs) as probes and using methanol-water as mobile phase. The investigations show that the new stationary phase behaves as a reversed phase stationary phase. The liquid chromatographic separation of PAHs solutes on the new bonded phase was compared with that on a p-tert-butyl-calix[4]arene-bonded silica stationary phase. The new p-tert-butyl-calix[8]arene-bonded phase exhibited higher retention and better separation selectivity, although the carbon content and coverage of the new packing material was lower than that of the p-tert-butyl-calix[4]arene bonded silica stationary phase. A possible retention mechanism for the new packing material was also proposed.     

Determination of Belotecan in the Plasma,Bile, and Urine of Rats by High-Performance Liquid Chromatography with Fluorescence Detection and Its Application to a Pharmacokinetic Study

Abstract

A simple and reliable high-performance liquid chromatographic (HPLC) method was developed for the determination of belotecan in the plasma, urine, and bile samples of rats. Belotecan was analyzed with HPLC using a C18 column with fluorescence detector. A mixture of acetonitrile–0.1 M potassium phosphate buffer at pH 2.4 (25:75, v/v) and 0.2% trifluoroacetic acid was used as the mobile phase. The lower limits of quantitation (LOQ) were 5 ng mL^?1 for the plasma and 5 µg mL^?1 for the urine and bile samples. The method has been readily applied for the routine pharmacokinetic study of belotecan in small laboratory animals.     

Evaluation of novel dendrimer chiral stationary phases using HPLC

Summary The reversed phase chromatographic properties of the [G1]-L-glutamic and ethyl ester-AC-silica (1), [G2]-L-glutamic acid ethyl ester-AC-silica (2) and the [G1]-L-glutamic acidt-butyl ester-AC-silica (3) dendrimer stationary phases were evaluated. Initial studies involved the comparison between these phases with a classic reversed phase (i.e. ODS1) by the separation of a standard reversed phase test mixture composed of dimethylphthalate, nitrobenzene, anisole, diphenylamine and fluorene. Separations were achieved with comparable performance to those obtained with the conventional reversed phase (ODS1). However, it was apparent that the chromatographic selectivity exhibited by the dendrimer stationary phases was different from that of the ODS1 phase. On a per mole basis, the dendrimers exhibited similar (and sometimes greater) affinity for these analytes compared with the ODS1 ligand. Subsequent chromatographic experiments were conducted upon the dendrimer chiral stationary phases using chiral analytes under reversed phase and normal phase conditions. Chiral resolution was not observed.     

Application of Thin-Layer Chromatography to High-Performance Liquid Chromatographic Separation of Steroidal Hormones and Cephalosporin Antibiotics

Abstract

High-performance liquid chromatographic (HPLC) separation of steroidal hormones and cephalosporin antibiotics was investigated by adsorption chromatography and reversed-phase chromatography, respectively.

Prior to the HPLC separation of these pharmaceuticals, silica gel thin-layer adsorption chromatography of steroidal hormones and reversed-phase thin-layer partition chromatography of cephalosporin antibiotics with chemically bonded dimethylsilyl silica gel were performed in order to obtain suitable HPLC separation systems.

In the separation of steroidal hormones, the same binary mobile phase ratios of TLC did not give satisfactory results in HPLC. For the sharp separation in HPLC, solvent strength in the binary solvent mixture used for TLC had to be decreased.

The difference in solvent strength for efficient separation between TLC and HPLC might be attributed to the fact that in HPLC the solvent elution power acts in an isocratic manner while in TLC it acts in a gradient manner.

On the other hand, a correlation of mobility between TLC and HPLC separation for cephalosporin antibiotics was obtained, and the possibility of direct transfer of chromatographic systems from TLC to HPLC for separation of these antibiotics was confirmed.     

Detection of gangliosides with the fluorochrome NBD dihexadecylamine and its application for preparative high performance thin layer chromatography.

A new method for the detection of gangliosides based on the lipophilic fluorescence agent 4-(N,N-dihexadecyl)amino-7-nitrobenz-2-oxa-1,3-diazole (NBD dihexadecylamine) and its application for preparative high performance thin layer chromatography is described. Brain gangliosides were chromatographed on silica gel coated thin layer plates and located with non-destructive fluorochrome under longwave ultraviolet light. The fluorescent zones were scraped off and the gangliosides were extracted with a mixture of chloroform/methanol/water (30/60/8; v/v/v). The gangliosides were separated from uncharged NBD dihexadecylamine by anion exchange chromatography and impurities were removed by Iatrobeads chromatography. The method described offers a simple and successful preparative thin layer chromatographic strategy to obtain pure gangliosides in microgram and milligram quantities.     

Separation of R(+)-and S(-)-Benzyl-3-Tetrahydrofuroates Using Two Different Chiral Columns

Abstract

A chiral separation of N(+)-and S(-)-benzyl-3-tetrahydrofuroate (I) and p-nitrobenzyl-3-tetrahydrofuroate (II) using a Chiralcel OB^© (cellulose tribenzoate) column with a hexane/2-propanol (60:40 v/v) mobile phase is described. Enantiomeric purity of R(+)-I was evaluated using the same chromatographic conditions. I was also separated using a Chiralspher^© (polyamides bonded to silica gel) column with an ethanol/distilled water (50:50 v/v) mobile phase.     

C18‐bound porous silica monolith particles as a low‐cost high‐performance liquid chromatography stationary phase with an excellent chromatographic performance

Ground porous silica monolith particles with an average particle size of 2.34 μm and large pores (363 Å) exhibiting excellent chromatographic performance have been synthesized on a relatively large scale by a sophisticated sol–gel procedure. The particle size distribution was rather broad, and the d(0.1)/d(0.9) ratio was 0.14. The resultant silica monolith particles were chemically modified with chlorodimethyloctadecylsilane and end‐capped with a mixture of hexamethyldisilazane and chlorotrimethylsilane. Very good separation efficiency (185 000/m) and chromatographic resolution were achieved when the C₁₈‐bound phase was evaluated for a test mixture of five benzene derivatives after packing in a stainless‐steel column (1.0 mm × 150 mm). The optimized elution conditions were found to be 70:30 v/v acetonitrile/water with 0.1% trifluoroacetic acid at a flow rate of 25 μL/min. The column was also evaluated for fast analysis at a flow rate of 100 μL/min, and all the five analytes were eluted within 3.5 min with reasonable efficiency (ca. 60 000/m) and resolution. The strategy of using particles with reduced particle size and large pores (363 Å) combined with C₁₈ modification in addition to partial‐monolithic architecture has resulted in a useful stationary phase (C₁₈‐bound silica monolith particles) of low production cost showing excellent chromatographic performance.     

Simultaneous Determination of Tinidazole and Furazolidone in Suspension by HPTLC and HPLC

Abstract

High performance thin layer chromatographic (HPTLC) and high performance liquid chromatographic (HPLC) methods were developed for the simultaneous determination of Tinidazole and Furazolidone in suspension.

In the HPTLC method the separation of Tinidazole and Furazolidone was carried out on silica gel 60F₂₅₄ HPTLC glass plate using chloroform:methanol:ammonia (9:1:0.1 v/v) as a mobile phase. Rf values obtained were 0.63 and 0.79 for Furazolidone and Tinidazole respectively. Densitometric evaluation was done at 335 nm. Linearity was obtained within the concentration range 10–50 μg/ml and 3.5–17.5 μg/ml for Tinidazole and Furazolidone respectively.

The second method is based on high performance liquid chromatography on a reversed phase column (μ Bondapak C₁₈) using a mobile phase comprised of water: acetonitrile: triethylamine (80:20:0.1 v/v) adjusted to pH = 3.0 with dil. phosphoric acid. Retention times were 5.24 and 7.82 min for Tinidazole and Furazolidone respectively at a flow rate of 1.5 ml/min. Detection was done at 335 nm. Linearity was obtained within the concentration range 30–180 μg/ml and 10.5–63 μg/ml for Tinidazole and Furazolidone resp.     

A study of the adsorption activities of silanol surface structures on a fused silica model substrate by combining 29Si CP MAS NMR and inverse gas chromatographic data

The possibilities of inverse gas-solid chromatography (IGC) in obtaining chromatographic data on fumed silica were examined. Aerosil A-200, a fused silica model substrate in ²⁹Si nuclear magnetic resonance analysis, was trimethylsilylated to different degrees. IGC was used to very reproducibly determine the free specific energies of adsorption of several functionalized probe solutes. Hydrogen bonding solutes have a free specific energy of adsorption that is at least about 50% higher than that of non-hydrogen bonding probe solutes. NMR was used in combination with elemental analysis to calculate surface concentrations of the different chemical surface structures. IGC data and surface concentrations were combined in order to determine the contribution of each type of surface structure to the total free specific adsorption energy. It could be concluded that residual silanols from the reaction of dihydroxydi-siloxysiloxane (Q₂ groups) with trimethylchlorosilane possess a higher adsorption activity than the silanols initially present.     

Enantiomeric recognition and separation of chiral organic ammonium salts by chiral pyridino-18-crown-6 ligands

Abstract

Optically pure allyloxy and dimethyl-substituted pyridino-18-crown-6 (8) was attached to silica gel by the following reactions. 4-Allyloxy-2,6-pyridinedimethyl ditosylate (23) was first prepared from chelidamic acid. Ditosylate 23 was treated with (S,S)-dimethyl-substituted tetraethylene glycol to form 8. Ligand 8 was treated with triethoxysilane using a platinum catalyst. The resulting chiral crown-substituted triethoxysilane 32 was reacted with silica gel in toluene at 90 C to attach the crown to silica gel. Preliminary results of the separation of [α-(1-naphthyl)ethyl]ammonium perchlorate into its (R) and (S) forms using the bound chiral crown with acetone/methanol (7/3) (v/v) as the eluant are reported. The preparation of chiral dimethyl(allyloxyphenyl)pyridino-18-crown-6 (9) that could be attached to silica gel on the side opposite to the pyridine ring is also reported.     

Development of an Isocratic SFC Method for Four Centrally Active Muscle Relaxant Drugs

Abstract

A reproducible and selective supercritical fluid chromatography (SFC) method was developed for the estimation of four centrally active muscle relaxants, viz., chlorzoxazone, methocarbamot, tizanidine, and baclofen, using guaifenesin (expectorant, muscle relaxant) as the internal standard. The effect of temperature, pressure and the modifier concentration on retention of the five solutes has been explored and the parameters optimised. An arbitrary mixture of 5 components was base line resolved on a JASCO- RP- C₁₈ (150 × 4.6 mm) 5μ metaphase column with a tertiary mobile phase of 9.1 % modifier [methanol containing 2 % glacial acetic acid, v/v] in carbon dioxide at 1.5 ml/min, 50°C temperature, and 12.75 MPa outlet pressure. UV detection at 235 nm was employed. Without the acetic acid in the mobile phase it was not possible to elute tizanidine and baclofen. With the additive, all the analytes produced symmetrical peaks. Quantitative validation concentration ranges have been found, and the performance data evaluated.     

配位色谱法从葛根素浸膏中分离纯化葛根素

建立了采用配位色谱柱从葛根素浸膏中分离纯化葛根素的方法。以铜离子为中心离子,制备了中心离子含量为7%的配位色谱柱。样品上样于配位色谱柱后,以氯仿-甲醇（体积比为10∶1）混合溶剂洗脱,得到了较纯的葛根素,较之用传统的硅胶色谱柱纯化,纯度提高了11%,回收率提高了12%,且柱容量提高了两倍。配位色谱改变了葛根素在传统硅胶柱上的洗脱顺序,对目标物质的分辨率比传统硅胶色谱柱高。     

Quantitation of Terfenadine,Pseudoephedrine Hydrochloride,and Ibuprofen in a Liquid Animal Dosing Formulation Using High Performance Liquid Chromatography

Abstract

Stability-indicating assay methods based on high performance liquid chromatography have been developed for the quantitation of terfenadine, pseudoephedrine hydrochloride, and ibuprofen when combined in an aqueous 0.5% w/v Tween 20 and 0.5% methylcellulose animal dosing formulation. Because of the diversity of this drug mixture two separate chromatographic systems were required for the assays. A reversed phase system using a 3-μm Spherisorb 0DS-2 column was used to assay for terfenadine and ibuprofen. An ion-exchange system using a 10-μm Partisil SCX column was used to assay for pseudoephedrine hydrochloride. The methods are accurate and precise with relative standard deviations over the concentration ranges of interest of 2% or less.     

Investigation of thin-layer chromatography properties of some transition metal complexes based on ditiocarbamates

Sodium diethyldithiocarbamate (DEDTC) and ammonium pyrolidinedithiocarbamate (PyDTC) are prepared as their Co or Cu (M) complexes [M(DEDTC)(2) and M(PyDTC)(2), respectively]. The complexes are prepared by reactions of DEDTC and PyDTC with metal (II) nitrates, and they are examined for chromatographic properties using thin-layer chromatography systems. These complexes and their mixtures are spotted on the activated and non-activated 250-microm thick thin layers of commercial silica gel (Si-60GF(254)). Pure toluene and a toluene-cyclohexane mixture (3:1, v/v) are used as mobile phases for running the complexes. These chromatographic systems are successfully used for the qualitative analysis of the corresponding metal cations and the separation of components in both M(DEDTC)(2) and M(PyDTC)(2) complex mixtures. In addition, non-activated layers are more successful than activated layers. This study may be useful in understanding the effects of stationary and mobile phase properties, retention mechanisms, and the effects of the nature of metal and ligand type on the chromatographic behavior and parameters [e.g., retention factors, theoretical plate numbers, and resolution] of the complexes.     

Platinum carbonyl derived catalysts on inorganic and organic supports: a comparative study

Fumed silica, silica gel, silica-alumina and cross-linked (5.5%) polystyrene have been functionalized with quaternary ammonium groups and the Chini cluster [Pt₁₂(CO)₂₄]²⁻ has been anchored onto these functionalized materials by ion pairing. A catalyst has also been prepared by the adsorption of Na₂[Pt₁₂(CO)₂₄] on unfunctionalized fumed silica. The catalytic activities of the resultant materials, and that of commercially purchased 5% platinum on alumina have been studied for the hydrogenation of a variety of unsaturated compounds. The substrates studied are: α-acetamidocinnamic acid, cyclohexanone, acetophenone, methyl pyruvate, ethyl acetoacetate, nitrobenzene and benzonitrile. Compared to the polystyrene supported catalyst, the inorganic oxide supported catalysts have higher surface areas and for most of the substrates have notably higher activities. The functionalized fumed silica-based catalyst gives higher conversions than functionalized silica gel and silica-alumina-based catalysts. In the hydrogenation of acetophenone and ethyl acetoacetate, the functionalized fumed silica-based catalyst show superior activity compared to the commercial platinum catalyst, and the catalyst made by conventional adsorption method. In benzonitrile hydrogenation with all the cluster-derived catalysts a hydrazine derivative is selectively formed, but when the commercial platinum catalyst is used benzyl amine is the main product.