Analysis of aromatic and terpenic constituents of pepper extracts by capillary electrochromatography

An original method based on CEC has been developed for the determination of aromatic and terpenic compounds in extracts of spices obtained from Piper nigrum. The method is based on the use of a fused silica capillary (effective length: 23.5 cm, internal diameter: 100 microm) packed with a C18 sorbent (packing length: 23 cm, particle size: 5 microm). The mobile phase is a 50 mM, pH 6.0 ammonium acetate/ACN (10:90 v/v) mixture. Applying a 30 kV voltage, the following 11 compounds were separated and analysed: terpinen-4-ol, caryophyllene oxide, limonene, alpha-pinene, 3-carene, beta-pinene, alpha-humulene, beta-caryophyllene, alpha-phellandrene, eugenol and piperine. Compound determination is carried out using a diode-array detector set at 265 and 338 nm for alpha-phellandrene and piperine, respectively, and at 210 nm (reference subtraction at 282 nm) for all the other analytes. The optimised method has been validated with good results in terms of linearity, limits of quantitation, detection and precision. The CEC method was successfully applied to the analysis of essential oils and methanolic extracts of 'black', 'white' and 'green' pepper.     

Capillary electrochromatography of methylated benzo[a]pyrene isomers. II. Effect of stationary phase tuning

For Part II of our ongoing study, we present a strategy for stationary phase optimization for the capillary electrochromatographic (CEC) separation of the 12 methylated benzo[a]pyrene (MBAP) isomers. Utilizing the optimum mobile phase conditions from Part I of our study as a guide, seven commercially available stationary phases have been evaluated for their ability to separate highly hydrophobic MBAP isomers. Ranging in design from high-performance liquid chromatography (HPLC) to CEC application, each phase was slurry packed in house and tested for CEC suitability and performance. Several stationary phase parameters were investigated for their effects on MBAP separation including bonding type (monomeric or polymeric, % carbon loading, surface coverage), pore size, particle size, and type of alkyl substituent. In this manner, the present state of commercially available packings has been assessed in our laboratory. Utilizing the optimum polymeric C18-5 microm-100 A-PAH stationary phase, the effects of CEC packed bed length and capillary inside diameter (I.D.) were also evaluated. A 50 microm I.D. capillary, 25 cm packed bed length and 75% (v/v) acetonitrile, 12.5 mM Tris, pH 8.0, 20 degrees C at 30 kV, provided resolution of 11 out of 12 MBAP isomers thus showing the effectiveness of CEC for analysis of structurally similar methylated polyaromatic hydrocarbons.     

In-line coupling of low-pressure short capillary electrochromatography columns to electrospray ionisation mass spectrometry

A new in-house designed and constructed injection valve for capillary electrochromatography (CEC) based on a rotating injection part with compartments for the eluent as well as for the sample has been coupled to a mass spectrometer via a sheath flow electrospray ionisation (ESI) interface, using short capillary columns of 15 cm length. The CEC columns were packed with 3 microm C(18) bonded silica particles, and a mixture of peptides was analysed using an ammonium acetate/acetonitrile eluent. A significant increase in the signal-to-noise ratio was obtained when the peptides were dissolved in water with the same content of organic modifier as in the eluent with an addition of 0.5% (v/v) acetic acid. When the CEC analysis was performed without any additional pressure, the separation current sometimes dropped tremendously due to bubble formation, caused by different permeability in the first and packed part of the column causing an extremely low electroosmotic flow. The separation current was restored to its original value by applying only 7 bar at the inlet of the CEC column, and the separation performance for the test peptides was recovered. A comparison of the CEC performance of peptides in pure CEC mode and in low-pressure CEC mode is reported.     

Method for simultaneous analysis of eight vitamin E isomers in various foods by high performance liquid chromatography and fluorescence detection

The objective of this study was to optimize a method to investigate the occurrence and to quantify the full isomeric composition of vitamin E (α-, β-, γ- and δ-tocopherols and tocotrienols) in 6 vegetables (raw and cooked), 3 herbs/spices, raw and cooked eggs, vegetable oils (canola, olive and soybean), flaxseed and sorghum (flour and seeds) and soy (flour) by HPLC with fluorescence detection. Different conditions of extraction and analysis were tested. The optimized method consisted of direct extraction with solvent (hexane:ethyl acetate, 85:15, v/v). For analysis normal phase column was used with mobile phase consisting of hexane:isopropanol:acetic acid (98.9:0.6:0.5) with isocratic elution and fluorescence detection. Excellent separation of all isomers was obtained along with adequate quantification in the foods analyzed. Recovery rates of standards ranged from 91.3 to 99.4%. The linearity range for each isomer varied from 2.5 to 137.5 ng/mL (R2 greater than 0.995 in all cases). Detection limits ranged from 21.0 to 48.0 ng/mL for tocopherols and from 56.0 to 67.0 ng/mL for tocotrienols, while quantification limits ranged from 105.0 to 240.0 ng/mL for tocopherols and from 280.0 to 335.0 ng/mL for tocotrienols. The optimized method was considered simple, fast and reliable, and also preserved vitamin E isomers when compared to validated methods involving saponification.     

Packing capilllary electrochromatography columns using vacuum--a preliminary study

This paper introducesa novel method for packing Capillary Electrochromatography Columns (CEC). Using vacuum packing methodology, silica particles as small as 1 microm were successfully packed into the capillary columns with 75 microm inner diameter. The columns are verystable and show no noticeable loss in efficiency after 200 sample injections. The performance of these vacuum packed capillary columns was evaluated with a mixture of aromatic and non-aromatic compounds. A 24 cm long capillary column can produce peak efficiencies of around 45,000 plates for benzene.     

Use of vancomycin silica stationary phase in packed capillary electrochromatography I. Enantiomer separation of basic compounds

Chiral separation of basic compounds was achieved by using 75 or 100 microm ID fused-silica capillaries packed with a vanoomycin-modified diol silica stationary phase. The capillary was firstly packed for about 12 cm with a slurry mixture composed of diolsilica (3:1) then with the vancomycin modified diol-silica (3:1) (23 cm), and finally with diol-silica (3:1) for about 2 cm. Frits were prepared by a heating wire at the two ends of the capillary; the detector window was prepared at 8.5 cm from the end of the capillary where vancomycin was not present. The influence of the mobile phase composition (pH and concentration, organic modifier type and concentration) on the velocity of the electroosmotic flow, chiral resolution and enantioselectivity was studied. Good enantiomeric resolution was achieved for atenolol, oxprenolol, propranolol, and venlafaxine using a mobile phase composition of 100 mM ammonium acetate solution (pH 6)/water/acetonitrile (5:5:90 v/v/v) while for terbutaline a mixture of 5:15:80 v/v/v provided the best separations. The use of methanol instead of acetonitrile caused a general increase of enantiomer resolution of the studied compounds together with a reduction of efficiency and detector response. However, the combination of acetonitrile and methanol in the mobile phase (as, e.g., 10% methanol and 80% acetonitrile) allowed to improve the enantiomer resolution with satisfactory detector response.     

Capillary electrochromatography using fibers as stationary phases.

Fiber-packed capillary columns have been evaluated in chromatographic performance in capillary electrochromatography (CEC). The change of electroosmotic flow (EOF) velocity and selectivity using different kinds of fiber materials was examined. Although the EOF velocity among the different fiber packed columns was almost the same, retention of parabens was larger on the Kevlar-packed column than on the Zylon-packed one, and was larger on the as-span-type fiber-packed column than on the high-modulus-type packed one. Using 200 microm ID x 5 cm Kevlar packed column combined with a 100 microm ID x 20 cm precolumn capillary and a 530 microm ID x 45 cm postcolumn capillary, the separation of three parabens within 30 s was achieved. Other compounds were also separated in a few minutes by the fiber-packed CEC method.     

Determination of tocopherols in vegetable oils by CEC using methacrylate ester-based monolithic columns

The separation and determination of tocopherols (Ts) in vegetable oils by CEC using methacrylate ester-based monolithic columns has been developed. The effects of pore size of the monolithic columns were studied, and the composition of mobile phase was optimized. The optimal pore size of the monolith was obtained with 12 wt% 1,4-butanediol in the polymerization mixture. Excellent resolution between tocopherols was achieved within 10 min analysis time with a 99:1 v/v MeOH-aqueous buffer containing 5 mM tris(hydroxymethyl)aminomethane at pH 8.0. The LODs were lower than 2.3 microg/mL, and interday and column-to-column reproducibilities at 25 microg/mL were better than 5.6%. Using a 93:7 v/v MeOH-aqueous buffer, both tocopherols and tocotrienols (T(3)s) of grapeseed and palm oils were resolved. Application to the detection of olive oil adulteration with low-cost edible oils was demonstrated.     

Capillary electrochromatography of 1-phenyl-3-methyl-5-pyrazolone derivatives of some mono-and disaccharides

Summary A packing procedure was adopted for capillary electrochromatography (CEC) that produces capillary columns with high separation efficiencies. The columns were fully packed, 50 cm long, with UV detection being performed through the packed section 30 cm from the inlet end. The CEC experiments were run at ambient pressure, with no additional pressure applied to the ends of the column. The stationary phase (octadecyl silica (ODS), 5 μm) promoted a high velocity electroosmotic flow (EOF), enabling rapid and efficient separation of a hydrocarbon test mixture. Some 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatives of mono- and disaccharides were baseline separated, using a 5 mM NaH₂PO₄ in 80% acetonitrile and 20% water (v/v) buffer solution. CEC shows promise for future applications in carbohydrate analysis. Presented at Balaton Symposium on High Performance Separation Methods, Siófok, Hungary, September 1–3, 1999     

Determination of major carotenoids in vegetables by capillary electrochromatography

A simple and rapid method for the isocratic separation and determination of carotenoids (carotenes and xanthophylls) in vegetables by CEC is described. The capillary column (100 microm ID, 25 cm effective length) was packed with 3 microm Hypersil ODS particles. The optimized mobile phase contained 60% ACN, 35% THF and 5% of a 5 mM Tris aqueous buffer of pH 8. beta-Carotene, lycopene and lutein were separated with efficiencies of 66 000-128 000 plates/m within a short time (less than 12 min for the last peak eluted, 13/13'-cis-beta-carotene). An excellent resolution of the three carotenoids, as well as partial resolution of their geometrical isomers, was achieved. Application to the determination of the analytes in carrot, tomato, spinach and corn was demonstrated.     

Analysis of selected withanolides in plant extract by capillary electrochromatography and microemulsion electrokinetic chromatography

Microemulsion electrokinetic chromatography (MEEKC) coupled with a diode-array detector was developed for the simultaneous analysis of natural steroidal compounds, withanolides including withaferin A, withacnistin and iochromolide. Optimal resolution was obtained with a microemulsion consisting of 70 mM octane, 800 mM 1-butanol, 100 mM sodium dodecyl sulfate (SDS), and 10 mM phosphate-borate buffer (pH 7) using a fused-silica capillary at 25 kV and 40 degrees C. Since this technique is not compatible with mass spectrometry detection, a capillary electrochromatographic method was developed to separate the investigated withanolides. The effects of mobile phase composition and pH were systematically investigated. Complete separation was obtained with a capillary electrochromatography (CEC) Hypersil C18 bonded silica column (packed length, 25 cmx100 microm ID and 375 microm OD), packed with 3 microm particles. The mobile phase consisted of formic acid-ammonia, pH 8 / acetonitrile (40/60 v/v); the voltage was set at 25 kV and the temperature at 20 degrees C. Under these conditions, resolution of these closely related compounds, including the critical pair withacnistin and iochromolide, was achieved in less than 5 min. The separations by MEEKC and CEC were compared with that obtained by reversed-phase liquid chromatography and showed similar retention order, indicating the analogy of the retention mechanism of these techniques. To further improve specificity and sensitivity, the developed CEC method was interfaced with electrospray ionization mass spectrometry using a Teflon connection between the CEC column and a void fused-silica capillary. Finally, the described methods were applied to the qualitative analysis of withanolides in Iochroma gesnerioides plant extract.     

Nonaqueous electrochromatography on C30 columns: separation of retinyl esters.

A nonaqueous packed capillary electrochromatographic reversed-phase method for separation of retinyl esters has been developed. The retinyl esters all-trans-retinyl acetate, palmitate, heptadecanoate, stearate, oleoate, and linoleoate were separated on a 180 microm ID column packed with 5 microm C30 particles with a mobile phase consisting of 2.5 mM lithium acetate in N,N-dimethylformamide-methanol (99:1, v/v). With this mobile phase, the electroosmotic flow was 0.8 mm/s at 650 V/cm and 40 degrees C, and the separation was completed in less than 30 min on 30 cm columns. To obtain electrostable frits of the hydrophobic C30 material both the preparation of the frits and the conditioning of the column prior to electroconditioning were of importance. Selectivity changes were introduced by replacing up to 70% v/v of the N,N-dimethylformamide by acetonitrile. The increase in the amount of acetonitrile was, however, accompanied by a significant increase in analysis time. Liver extracts obtained from arctic seal were analyzed.     

Acrylate ester-based monolithic columns for capillary electrochromatography separation of triacylglycerols in vegetable oils

A simple and reliable method for the evaluation of triacylglycerols (TAGs) in vegetable oils by capillary electrochromatography (CEC) with UV-Vis detection, using octadecyl acrylate (ODA) ester-based monolithic columns, has been developed. The percentages of the porogenic solvents in the polymerization mixture, and the mobile phase composition, were optimized. The optimum monolith was obtained at the following ratios: 40:60% (wt/wt) monomers/porogens, 60:40% (wt/wt) ODA/1,3-butanediol diacrylate and 23:77% (wt/wt) 1,4-butanediol/1-propanol (14 wt% 1,4-butanediol in the polymerization mixture). A satisfactory resolution between TAGs was achieved in less than 12 min with a 65:35 (v/v) acetonitrile/2-propanol mixture containing 5 mM ammonium acetate. The method was applied to the analysis of TAGs of vegetable oil samples. Using linear discriminant analysis of the CEC TAG profiles, the vegetable oils belonging to six different botanical origins (corn, extra virgin olive, hazelnut, peanut, soybean and sunflower) were correctly classified with an excellent resolution among all the categories.     

Use of nano-liquid chromatography for the analysis of glycyrrhizin and glycyrrhetic acid in licorice roots and candies

Glycyrrhizin (G) and glycyrrhetic acid (GA) were separated by using nano-liquid chromatography (nano-LC) in a fused silica capillary packed with RP18 stationary phase (75 microm ID, effective length 33 cm, packed 23 cm) eluting at 300 nL/min in a gradient mode. The mobile phase was a mixture of water-MeOH-MeCN-acetic acid (29:35:35:1, v/v/v/v) that was delivered for one minute and after this was modified by reducing the water content (14:42.5:42.5:1, v/v/v/v). The intra-day and inter-day relative standard deviations (of retention time and peak area) were satisfactory (lower than 2.9 and 4%, respectively). The linearity of the nano-LC method was assessed in the range 0.62-5.00 microg/mL and 80-200 microg/mL for GA and G, with R2 = 0.996 and 0.995, respectively. The licorice was extracted with a mixture of ethanol-water, diluted with the mobile phase, and injected for the analysis.     

Reversed-phase capillary electrochromatography for the simultaneous determination of acetylsalicylic acid, paracetamol, and caffeine in analgesic tablets

The separation and simultaneous determination of caffeine, paracetamol, and acetylsalicylic acid in two analgesic tablet formulations was investigated by capillary electrochromatography (CEC). The effect of mobile phase composition on the separation and peak efficiency of the three analytes was studied and evaluated; in particular, the influence of buffer type, buffer pH, and acetonitrile content of the mobile phase was investigated. The analyses were carried out under optimized separation conditions, using a full-packed silica capillary (75 microm ID; 30.0 cm and 21.5 cm total and effective lengths, respectively) with a 5 microm C8 stationary phase. A mixture of 25 mM ammonium formate at pH 3.0 and acetonitrile (30:70 v/v) was used as the mobile phase. UV detection was at 210 nm. Good linearity was found in the range of 50-200, 20-160, and 4-20 microg/mL for acetylsalicylic acid (r2=0.9988), paracetamol (r2=0.9990) and caffeine (r2=0.9990), respectively. Intermediate precision (RSD interday) as low as 0.1-0.8% was found for retention times, while the RSD values for the peak area ratios (Aanalyte/AIS) were in the range of 1.9-2.9%. The optimized CEC method was applied to the analysis of the studied compounds present in commercial tablets.     

Capillary electrochromatography/nanoelectrospray mass spectrometry for attomole characterization of peptides

The successful coupling of capillary electrochromatography (CEC) to an ion trap mass spectrometer via a nanoelectrospray interface (nESI) is described. Using a conductively coated tip butted to the end of a CEC column, it was possible to obtain a stable spray without any sheath liquid being employed. Selected small peptides were separated with CEC columns (100 microm i.d./25 cm long) packed with 3 microm Hypersil C8 or C18 bonded silica particles with an eluent composed of ammonium acetate/acetonitrile. Peptide mixtures of desmopressin, peptide A, oxytocin, carbetocin and [Met(5)]-enkephalin were detected in the mid-attomole range, which is the lowest amount analyzed using CEC combined with MS detection. It was also observed that sensitivity can be compromised at higher separation voltages. We demonstrate that CEC/nESI-MS, at the current stage of development, represents one of the most sensitive systems for peptide analysis.     

Life-time studies with capillary electrochromatography columns operated under different conditions

A test system has been established to permit the monitoring of the life-time performance of several reversed- phase capillary electrochromatography (CEC) columns. The retention factors, k(cec), peak symmetry coefficients, lambda(sym), and column efficiencies, N, of three neutral n-alkylbenzene analytes, namely ethyl-, n-butyl- and n-pentylbenzenes, were determined for Hypersil 3 microm n-octylsilica and n-octadecylsilica packed into CEC capillary columns of 100 microm I.D., with a packed length of 250 mm, and a total length of 335 mm. The performances of these CEC capillary columns were examined for a variety of eluents with pH values ranging between pH 2.0 - 8.0, similar to those employed to study the retention behaviour of peptides that we have previously reported. The relative standard deviation (RSD) of the retention factors (k(cec) values) of these n-alkylbenzenes, acquired with an eluent of (25 mM Tris-HCl, pH 8.0,)-acetonitrile (1:4, v/v), when the CEC capillary columns were used for the first time (virgin values), were 4% (based on data acquired with 4 CEC capillary columns) for the n-octyl bonded silica capillary columns, and 6% (based on 8 columns) for n-octadecyl bonded silica capillary columns. The RSD values of the k(cec) values of the n-alkylbenzenes for one set of replicates (n=6) with one CEC capillary column was < 0.5%. The theoretical plate numbers, N, for the virgin CEC capillary columns were ca. 60,000, whilst the observed N values for all new CEC capillary columns were > or = 40,000 for n-octyl bonded silica capillary columns and > or = 50,000 for n-octadecyl bonded silica capillary columns. The peak symmetry coefficients, lambda(sym), of the n-alkylbenzenes for virgin CEC capillary columns and for CEC capillary columns used for more than 1,000 injections were always in the range 0.95-1.05. The experimental results clearly document that the life-time performance of the CEC capillary columns depends on the eluent composition, as well as the nature of the analytes to which the CEC capillary columns are exposed.     

Rapid separation of barbiturates and benzodiazepines by capillary electrochromatography with 3-(1,8-naphthalimido)propyl-modified silyl silica gel

A capillary electrochromatographic (CEC) method was applied to the simultaneous separation of barbiturates (barbital, phenobarbital, secobarbital and thiopental) and benzodiazepines (nitrazepam, diazepam and triazolam). The separation was performed in a 75 microm i.d. capillary, packed with 3-(1,8-naphthalimido)propyl-modified silyl silica gel (NAIP), studying the effects of buffer pH and mobile phase composition. Using an applied voltage of 20 kV and the short-end injection method (9 cm capillary effective length), the mobile phase of 1.0 mM citrate buffer (pH 5.0) containing 45% methanol provided the baseline separation of seven toxic drugs in less than 9 min. In CEC with NAIP, the benzodiazepines were separated by the combination of hydrophobic and pi-pi interactions, whereas the separation of the barbiturates was based on the hydrophobic interaction.     

毛细管反相电色谱法分离行为的研究

对乙睛-水-磷酸二氢销体系毛细管反相电色谱分离行为进行了研究。采用柱上紫外检测,在75μmi.d.×30cm的毛细管ODS（3μm）填充柱上获得了小于2.0的折合培板高度。同时还研究了乙睛的比例、电解质的浓度和电场强度等因素对电渗流和往效的影响。     

Separation of the insecticidal pyrethrin esters by capillary electrochromatography

A simple, rapid technique for the direct separation and quantification of the six insecticidally active pyrethrin esters in typical extracts and commercial formulations by capillary electrochromatography (CEC) has been described. The separation of the pyrethrin esters was achieved by optimizing several parameters including the length of stationary phase, the mobile phase composition and column temperature. The mobile phase composition had the most pronounced effect toward resolving these structurally similar compounds. A ternary mobile phase composed of acetonitrile-aqueous buffer-tetrahydrofuran (55:35:10) provided the elutropic solvent strength needed to resolve the six esters from an extract mixture in under 16 min. A 25 cm packed bed of Hypersil 3 microm C18 stationary phase was used with the ternary mobile phase at 25 degrees C and 30 kV voltage. These conditions also yielded excellent separation of the pyrethrin esters in two different commercially available insecticidal formulations. In addition, the developed CEC method was shown to be a fast and easy way of quantifying the amount of these esters in typical pyrethrin formulations.