Separation of stilbenes by capillary electrophoresis and high-performance liquid chromatography

Stilbenes, fluorescence whitening agents (FWAs), are usually added to cleaning agents in household and in industry. Capillary electrophoresis (CE) was often applied to separate various compounds simultaneously for its multinomial advantages. In this paper, we established analytical methods of six diaminostilbenes with CE and ion-pair chromatography (IPC). The optimum mobile phase for IPC was 11.78 mM tetrabutylammonium hydrogen sulfate (TBA) aqueous and acetonitrile. An IPC method has been developed for simple and direct separation for diaminostilbenes, anionic substances, with TBA as ion-pair reagent. Satisfactory linear ranges (7.0 x 10(-3) approximately 3.0 x 10 microg/mL), correlation coefficients (0.9992-0.9999), and detection limits (6-13 ng/mL) were obtained. Separations were also performed by capillary zone electrophoresis (CZE) using a buffer consisting of Tris (pH 10.1), n-tetradecyltrimethylammonium bromide (TTAB) and acetonitrile. A linear range of 5.0 x 10(-1) - 4.0 x 10 microg/mL, correlation coefficients between 0.9975 and 0.9998, and detection limits between 337 and 446 ng/mL were obtained. In particular, the separation of a pair of similar compounds (mass difference of 2) was achieved by addition of TTAB. The optimum analytical methods of CE and high-performance liquid chromatography (HPLC) were applied to commercial household with direct analysis and standard addition. No significant bias were shown between them by t-test at 95% confidence level.     

Separation mechanism and determination of flavanones with capillary electrophoresis and high-performance liquid chromatography

To probe separation mechanism and determination with capillary zone electrophoresis (CZE) and liquid chromatography (LC), nine compounds with identical flavanone skeleton were studied. Optimum separation of LC was attained with gradient of acetonitrile and 5mM phosphate buffer (pH 6.9). For CE, electrolyte buffer was 4.5mM SDS in 32mM sodium tetraborate buffer (pH 9.2). The distinguishing feature in this work was successful separation of monohydroxyl stereoisomers by CZE. Polarity is generally increased with hydroxyl groups. In a separation mechanism study, polarity would be reduced by intramolecular hydrogen bond between hydroxyl of C5 and carbonyl group of C4. Comparison of the retention results among monohydroxyl flavanones shows polarity with hydroxyl at C6 the least, and that at C4' and C7 nearly equal. Also, elution order of flavones and flavanones would be adverse due to the hydroxyl at C3 in LC. From the numerical value pK(a) of flavanone, the C7-OH is the smallest, and two hydroxyl groups in an adjacent position is always less than the unique one caused by forming a stable 5-membered ring. Investigation of separation mechanism yield only the effect of constituent but also reasonable explanation for contradictory results between Wulf and our laboratory, this due to the hydroxyl at C3.     

Determination of flavonoids by high-performance liquid chromatography and capillary electrophoresis

The compounds of flavonoid, an important group in nature, can prevent coronary heart disease and anticancer by virtue of the characteristics of antioxidation. Nine flavonoids most often seen in grape wine, namely apigenin, baicalein, naringenin, luteolin, hesperetin, galangin, kaempferol, quercetin, and myricetine, were determined by means of high-performance liquid chromatography (HPLC) and capillary zone electrophoresis (CZE) in this work. A successful resolution was obtained from an unusual additive of tetrahydrofuran in mobile phase by HPLC. One notable thing is that the mixture of luteolin and quercetin could be separated for the first time by HPLC. In addition, the better detection limit was still attainable even with the use of tetrahydrofuran. The detection limits of CZE performed in borate buffer were hundreds-fold better than in previous reports. Furthermore, the retention and migration behavior of the analytes studied were discussed. As the result of this study, the elution order of flavone and flavonone was reversed to the contention proposed by Wulf et al. It was predictable from the interaction with tetrahydrofuran. Consequently, the extracts from grape wine with solid-phase extraction were analyzed by developing methods of HPLC and CZE. The obtained recoveries ranged from 90 to 107% and the relative standard deviations were under 6.3%.     

Analysis of tea components by high-performance liquid chromatography and high-performance capillary electrophoresis

Tea is one of the most popular beverages in the world. The number of reports on the analysis of tea components, especially for catechins, has recently been increasing. We review the recent reports on the analysis of tea components using the analytical methods of high-performance liquid chromatography and high-performance capillary electrophoresis.     

Quantitation of insulin injection by high-performance liquid chromatography and high-performance capillary electrophoresis.

High-performance capillary electrophoresis (HPCE) was evaluated as a potential technique for the regulatory analysis of commercial dosage forms of insulin. A comparison was made to a liquid chromatographic analysis presently being proposed as an official monograph in the United States Pharmacopeia. The salient points of this comparison were accuracy, precision and ease of use. Both authentic (i.e. single blind, spiked) samples and commercial pharmaceutical formulations (injections) were examined. Chromatographic analyses of both commercial formulations and authentic samples were characterized by good precision, with accuracy being supported by results from authentic (spiked) samples. Conventional HPCE (by which is meant a non-micellar electrolyte used with an uncoated, unmodified fused-silica capillary) achieved reasonable accuracy, but less than impressive precision, when applied to authentic samples. When used for commercial formulations, this type of HPCE did not produce a level of accuracy suitable for regulatory purposes, even with the use of an internal standard.     

Analysis of carbohydrates in glycoproteins by high-performance liquid chromatography and high-performance capillary electrophoresis

We describe two methods for the analysis of oligosaccharide chains in glycoproteins by high-performance liquid chromatography (HPLC) and high-performance capillary electrophoresis (HPCE).O-andN-glycosidically linked oligosaccharides released from glycoproteins can be identified as their borohydride-reduced forms by anion-exchange HPLC with pulsed amperometric detection.N-Glycosidically linked oligosaccharides can also be analyzed as 2-aminopyridine derivatives by HPCE in direct zone electrophoresis mode in an acidic phosphate buffer and zone electrophoresis mode as borate complexes in an alkaline buffer. We also present a convenient procedure for the analysis of the constituent monosaccharides of these oligosaccharides chains by HPLC based on reversed-phase partition mode as 1-phenyl-3-methyl-5-pyrazolone derivatives.     

Characterization of mixtures of biocidal oligoguanidines by capillary electrophoresis and high-performance liquid chromatography coupled to mass spectrometry

The polycondensation of guanidine hydrochloride and 2,2′-(ethylenedioxy)bis(ethylamine) leads to various types of oligomeric guanidines exhibiting a broad spectrum of biocidal activities. In the present work a reversed-phase high-performance liquid chromatographic method with a gradient consisting of aqueous 0.1% trifluoroacetic acid and acetonitrile as mobile phase has been developed to separate these oligoguanidines according to type and chain length. The combination with electrospray mass spectrometry allowed the identification of the various compounds. By this technique, some structures already suggested in the literature could be confirmed, and several additional oligoguanidines not yet reported could be identified. As a complementary technique, capillary zone electrophoresis was investigated. Best results were obtained with carrier electrolytes consisting of phosphoric acid in water/acetonitrile mixtures. Although the number of peaks that could be separated by the electrophoretic method was considerably lower than in case of the chromatographic method, capillary electrophoresis in combination with UV detection at 195 nm may still be a fast method suitable for quantitation of some of the major compounds and for monitoring the reaction rate during the polycondensation reaction.     

Separation of capsular polysaccharide K4 and defructosylated K4 derived disaccharides by high-performance capillary electrophoresis and high-performance liquid chromatography

A rapid, highly sensitive and reproducible high-performance capillary electrophoresis (HPCE) method (electrokinetic chromatography with sodium dodecyl sulfate) is described for the determination of disaccharides present in the polysaccharide from the uropathogenic Escherichia coli K4 bacteria (05:K4:H4) and its defructosylated product. Following chondroitinase digestion of K4 and its derivative, the two disaccharides, DeltaHexAFrc-GalNAc for K4 and deltaHexA-GalNAc for defructosylated K4, are separated and readily determined within 20 min on an uncoated fused-silica capillary using normal polarity at 20 kV and detection at 230 nm. Comparison was made by separation of these two disaccharides in isocratic strong-anion exchange HPLC. A linear relationship was found for the two unsaturated disaccharides over a wide range of concentrations, from approximately 0.5 to 5 micro g for high-performance liquid chromatography (HPLC) and from approximately 0.06 to 0.3 micro g for HPCE. The HPCE separation produced a greater detection sensitivity (about 10 times greater) than HPLC. The described methods were used to evaluate the defructosylation process of K4 under drastic acid conditions. Good correspondence was found for the amount of unsaturated disaccharides for the two techniques.     

Determination of linear alkylbenzenesulfonates by high-performance liquid chromatography and capillary zone electrophoresis

High-performance liquid chromatography and capillary zone electrophoresis have been used for the separation of homologues and structural isomers of linear alkylbenzenesulfonates with subsequent UV detection. The analytical advantages of these techniques are applied to the analysis of technical products containing high amounts of LAS as well as river water samples with very low LAS concentrations using preconcentration steps.Dedicated to Professor Dr. H. Kriegsmann on the occasion of his 70th birthday     

Comparative analysis of tea catechins and theaflavins by high-performance liquid chromatography and capillary electrophoresis

This paper describes the simultaneous determination of catechins and theaflavins in green and black teas, using reversed-phase high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE). The tea polyphenols analyzed included (+)-catechin, catechin gallate, (-)-epicatechin, epicatechin-3-gallate, epigallocatechin, epigallocatechin-3-gallate, theaflavin, theaflavin-3-monogallate, theaflavin-3'-monogallate and theaflavin-3,3'-gallate. These polyphenols together with six other tea ingredients such as caffeine, adenine, theophylline, quercetin, gallic acid and caffeic acid were separated within 27 min by HPLC and in less than 10 min by CE. The optimal analytical conditions of both chromatographic methods were investigated for the convenience and reliability for routine analysis. Both HPLC and CE were found to be reliable and compatible. The reproducibility of the within-day assay using both methods was generally >90%. The day-to-day variation of retention time was <5% for HPLC, while the variation of migration time for CE was <2%. The analysis time of CE was three-times faster, however it is five-times less sensitive than HPLC, which has detection limits of 0.05 microg/ml and 0.5 microg/ml for catechins and theaflavins, respectively.     

Comprehensive two-dimensional separations based on capillary high-performance liquid chromatography and microchip electrophoresis

A novel comprehensive two-dimensional (2-D) separation system coupling capillary high-performance liquid chromatography (cHPLC) with microchip electrophoresis (chip CE) is demonstrated. Reversed-phase cHPLC was used as the first dimension, and chip CE acted as the second dimension to perform fast sample transfers and separations. A valve-free gating interface was devised simply by inserting the outlet-end of LC column into the cross-channel on a specially designed chip. A home-made confocal laser-induced fluorescence detector was used to perform on-chip high-sensitive detection. The cHPLC effluents were continuously delivered to the chip and pinched injections of the effluents every 20 seconds were employed for chip CE separation. Gradient elution of cHPLC was carried out to obtain the high-efficiency separation. Free-zone electrophoresis was performed with triethylamine buffer to achieve high-speed separation and prevent sample adsorption. Such a simple-made comprehensive system was proved to be effective. The relative standard deviations for migration time and peak height of rhodamine B in 150 sample transfers were 3.2% and 9.8%, respectively. Peptides of the fluorescein isothiocyanate (FITC)-labeled tryptic digests of bovine serum albumin were fairly resolved and detected with this comprehensive 2-D system.     

Pesticides analysis by liquid chromatography and capillary electrophoresis

Nowadays, a wide range of pesticides are used in agricultural production, and their monitoring in samples of environmental and alimentary interest is of extreme importance to ensure, among others, the safety of consumption of foods. The aim of this work is to provide updated information about the major developments in CE and HPLC in pesticide analysis, covering relevant publications between 2004 and early 2006. The use of different sample pretreatment steps to provide a suitable extraction of these compounds from the different matrices as well as to increase the sensitivity of the determination is also discussed.     

Separation of dideoxyribonucleosides in trace amounts by automated liquid chromatography and capillary electrophoresis.

No satisfactory high-performance liquid chromatographic (HPLC) method is currently available for the separation of the major dideoxyribonucleosides (ddNs) and their derivatives. A method involving HPLC has been developed for the separation of five major ddNs [ddA, ddC, ddI, azT and 2',3'-dideoxy-2',3'-didehydrothymidine (d4T)]. Elution of the common and modified components of DNA was also examined under the selected separation conditions of HPLC. The elution characteristics of these compounds were studied using serum plasma samples spiked with ddN derivatives. In addition, capillary electrophoresis (CE) was investigated for the separation of ddNs and their derivatives. Picomolar amounts of the five major ddNs and the metabolic product of azT [5'-O-glucuronide-3'-azido-3'-deoxythymidine (Glo-azT)] were satisfactorily resolved in 10 min by using a modification of CE. The spectral properties of the ddNs were characterized under different pH conditions and compared with those of their parent deoxyribonucleosides (dNs) because these compounds are commonly measured in HPLC by their spectral properties. The spectra of ddC and ddT derivatives resemble very closely those of dC and dT, but those of ddA and ddI differ to some extent from their parent dNs. The HPLC method was extensively examined for satisfactory resolutions of these compounds. For example, an isocratic elution method, although simple, failed to resolve these compounds and ion-pair chromatography did not offer any advantage. Gradient elution involving buffered solutions and increasing amounts of an organic modifier yielded satisfactory results. Methanol appeared to be the organic modifier of choice. A reversed-phase matrix with smaller than octadecyl alkyl chains did not produce the necessary interactions. Uniform spherical beads of smaller diameter produced superior resolutions. The separation of these compounds on three commercially available columns is discussed. The separation of human plasma samples spiked with dideoxynucleoside derivatives by HPLC was accomplished in ca. 16 min. The presence of the dNs did not interfere in their separations.     

Determination of Triton X-100 in influenza vaccine by high-performance liquid chromatography and capillary electrophoresis

Triton X-100 is applied to influenza vaccines at different stages of the manufacturing process to prevent aggregation and precipitation of biomolecules. Furthermore it is used to disintegrate the virus particles in split vaccine and to guarantee the homogeneity during production and utilisation. The final concentration of Triton X-100 has to be determined because the concentration changes in manufacturing process. The determination of the total amount of Triton X-100 as well as the separation of its ethylene oxide oligomers was possible with high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE). In HPLC a change of the column and eluent was necessary, in CE different electrolytes were used for the various separation effects. The HPLC method for the analysis of total Triton was preferred for the quantification of Triton X-100 in influenza vaccine because of better linearity, reproducibility and detection sensitivity compared to CE. In the end products an average concentration of 0.117 mg/mL was found. Received: 19 December 1996 / Revised: 27 February 1997 / Accepted: 6 March 1997     

High performance separations of nucleic acids using capillary electrophoresis and high-performance liquid chromatography

High resolution separations of nucleic acids have been performed using high performance capillary electrophoresis (HPCE) and high performance liquid chromatography (HPLC). Electropherograms showing HPCE separations of single and double stranded DNA are presented and compared with HPLC separations. Single base resolution of poly(dA) oligonucleotides in the size range of 12 to 60-mers was achieved in 35 min using HPCE. Plate numbers for HPCE are in the hundreds of thousands and reproducibility is about 1–2 % (RSD). In comparison with HPLC separations, the resolution of nucleic acids obtained using HPCE is much better than that using HPLC, while reproducibility of HPCE is comparable with that of HPLC.     

Separation of porphyrin isomers by high-performance liquid chromatography

A high-speed reversed-phase high-performance liquid chromatographic method using an octadecylsilyl 3 cm long (3 microns particle size) column to separate the free acids of uroporphyrins I and III and coproporphyrins I and III from each other, and from the type I isomers of several other porphyrin carboxylic acids, is described. Separation of the porphyrins was achieved in less than 8 min, and injections were possible every 12 min. The detection limits of uroporphyrin, coproporphyrin, and mesoporphyrin were 75, 45, and 35 fmol (at a signal-to-noise ratio of 2), respectively. Application of the method to the determination of urinary and liver porphyrin patterns is shown.     

Cross validation of capillary electrophoresis and high-performance liquid chromatography for cefotaxime and related impurities

Summary A micellar electrokinetic chromatography method is presented which permits separation of cefotaxime and its major related impurities. Separation was carried out at 15 kV, using 30 mM sodium dihydrogen phosphate adjusted to pH 7.2 with 5 M NaOH and which contained 165 mM sodium dodecylsulfate as electrolyte. Results obtained by capillary electrophoresis were in good agreement with those of high-performance liquid chromatography with respect to the level of the major known impurities, total impurity content and cefotaxime purity.     

Insoluble eggshell matrix proteins--their peptide mapping and partial characterization by capillary electrophoresis and high-performance liquid chromatography

Avian eggshell matrix proteins were studied by two analytical approaches. Peptide mapping was done by trypsin and pepsin followed by collagenase cleavage; analyses were carried out by capillary electrophoresis and reversed-phase high-performance liquid chromatography (HPLC). Comparison of peptide maps obtained by both methods revealed a complex mixture of peptides in the insoluble layers of the eggshell; it was concluded that there are at least three different insoluble protein/peptide layers in the avian eggshell (cuticle, palisade, and mammillary layer). Partial characterization of peptides in each layer was made by HPLC-mass spectrometry analysis. There is an evidence that the eggshell insoluble proteins contain species susceptible to collagenase cleavage, however, the sequences split by this enzyme probably are not those typical for the main triple-helical core of collagenous proteins. It is proposed that the action of collagenase upon eggshell proteins is caused by the side effect of collagenase described previously with synthetic peptides. Some of the proteins present are probably glycosylated. Fatty acid content in the insoluble eggshell layers (after decalcification) was in the range of 2-4% (which reflected both lipid and lipoproteins bound fatty acids). Porphyrin pigments are dominant in the cuticle layer.     

Comparison of high-performance liquid chromatography and high-performance capillary electrophoresis for the determination of cicletanine in plasma.

A sensitive and selective high-performance capillary electrophoresis (HPCE) procedure was developed for the determination of total cicletanine in human plasma. The procedure consisted in extraction of the drug with diethyl ether and analysis by micellar electrokinetic capillary chromatography in a fused-silica capillary using sodium dodecyl sulphate in the run buffers and ultraviolet detection. The concentrations of cicletanine obtained by this method were compared with those obtained by a high-performance liquid chromatographic (HPLC) method used routinely. The within-run precision of the methods, expressed as relative standard deviation, ranged from 1.6 to 7.8% for HPLC and from 6.4 to 11.1% for HPCE. Both methods showed an adequate level of accuracy; the relative errors ranged from 0.02 to 3.25% for HPLC and from 0.21 to 2.90% for HPCE. The HPCE method required less than half the time taken by the HPLC method, making HPCE a useful alternative technique for the routine determination of cicletanine in plasma. Both methods were used to follow the time course of total cicletanine in human plasma after a single oral therapeutic dose of the drug.     

Separation of natural and synthetic heparin fragments by high-performance capillary electrophoresis.

The application of capillary electrophoresis (CE) for the analysis of natural and synthetic low-molecular-mass heparin fragments at low pH is described. It is demonstrated that under the applied conditions the separation is based on charge, charge distribution and molecular mass of the heparin molecules, yielding a high resolution. It is shown that the presence of sodium chloride in the sample solution has hardly any effect on the CE performance. However, the pH of the electrophoresis buffer is a critical parameter. The resolutions obtained with CE and high-performance anion-exchange chromatography (HPAEC) are compared for various heparin fragments and it is concluded that, at least for this type of molecule, CE forms an attractive alternative to HPAEC.