Gradient chromatography isocratic conditions from

Numerous methods for high-performance Uquid chromatography (HPLC) of tropane alkaloids have been developed. The chromatographic conditions depend on the variability of the analyzed matrices (extracts from different plant tissues, pharmaceutical preparations, clinical, and forensic probes) and analytes (pure compounds or alkaloid mixtures with different composition). Most often, columns packed with reverse-phase Cl 8 stationary phase are used for the separation of tropane alkaloids. Gradient or isocratic elution generally involves buffered mixtures at the acidic pH of water—acetonitrile or acetonitrile—methanol, such as acetonitrile-triethylammonium phosphate buffer (25 75) at pH 6.2 [65] acetonitrile-50 mM phosphate buffer at pH 2.95 (10 90 and 20 80) [66], methanol-0.05 M... 

Size-exclusion chromatography combined with RP-HPLC-MS was employed for the separation of pyranoanthocyanins from red wine. Wine samples (10 ml) were acidified with 3 M HC1 to pH 1 then sodium bisulphite was added at a concentration of 400 g/1. After 15 min reaction time the treated wine was loaded into a gel column (200 X 15 mm i.d.). Pigments were eluted with 95 per cent ethanol followed with 100 per cent methanol. The various fractions were acidified to pH 1, concentrated and redissolved in water. HPLC-DAD was carried out in an ODS column (150 X 4.6 mm i.d. particle size 5 /nn) at 35°C. Solvents were 0.1 per cent aqueous TFA (A) and ACN (B). The gradient started with 10 per cent B for 5 min to 15 per cent B for 15 min isocratic for 5 min to 18 per cent B for 5 min to 35 per cent B for 20 min. The flow rate was 0.5 ml/min and analytes were detected at 520 nm. MS conditions were sheath and auxiliary gas were a mixture of nitrogen and... 

Figure 8 Affinity chromatography for the analysis of the methanol extract of Artemisia capillaris under optimized conditions. SCO, scoparone CAP, capillarisin. Experimental conditions the column used was of 150 mm x 4.6 mm ID packed with HSA immobilized on silica (7 pm), column temperature was 35 °C, flow rate was 0.8ml min-1, and UV detection wavelength was set to 238 nm. Initially, 10 min isocratic elution with mobile phase of 1.5% acetonitrile in 10 mmol I-1 phosphate buffer (pH 6.0) then, 5 min linear gradient elution from 1.5 to 12% acetonitrile in 10 mmol I-1 phosphate buffer (pH 6.0) with the elution of the latter mobile phase kept for an additional 45 min and finally, another 45 min linear gradient elution from 12% acetonitrile in 10 mmol I-1 phosphate buffer (pH 6.0) to 15% acetonitrile in 10 mmol I-1 phosphate buffer (pH 7.4). Reproduced from H. L. Wang H. F. Zou J. Y. Ni L. Kong S. Gao B. C. Guo, J. Chromatogr. A 2000, 870, 501-510.

Figure 1 Separations on silica IDA columns with different elution protocols. (A) Isocratic separation of four metal ions on a 100mmX4mm column packed with 5pm IDA silica. Eluent, lOmmoll nitric acid. Detection, PAR postcolumn reaction at 510nm. (Unpublished work, Nesterenko PN and Jones P.) (B) Isocratic separation of five metal ions on a 250 mm x 4 mm column packed with 5 pm IDA silica. Eluent, 0.5 mol I KCI, 20mmoll picolinic acid, and 12.5mmoll" nitric acid. Detection, PAR postcolumn reaction at 510 nm. (Unpublished work, Nesterenko PN and Jones P.) (C) Step gradient separation of Mn(ll), Cd(ll), Co(ll), Zn(ll), and Pb(ll). Eluent conditions 0.1 mol r NaCI (pH 2.6) switched to 0.1 mol 1 NaCI (pH 1.6) at time = 3 min prior to standard injection. Column, 250mm X 4 mm, packed with 8 pm silica IDA. Detection, PAR postcolumn reaction at 495 nm. (Reprinted with permission from Bashir W and Pauli B (2002) Ionic strength, pH and temperature effects upon selectivity for transition and heavy metal ions when using chelation ion chromatography with an iminodiacetic acid bonded silica get column and simple eluents. Journal of Chromatography 942 73-82 Elsevier.)...

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