High-performance liquid chromatography isocratic elution

Procedure Flavonoids are then further purified with 2 ml of methanolic HC1 (2 N), followed by centrifugation (2 min, 15 600 g), hydrolyzation of 150 il of suspension in an autoclave (15 min, 120 C). A reverse osmosis-Millipore UF Plus water purification system is used in high performance liquid chromatography (HPLC) with an autosampler. After injections of 5 pg of samples, the mobile phases flow at a rate of 1 ml/minute with isocratic elution in a column at 30 C. 

Product ester ee was determined by isocratic normal-phase high-performance liquid chromatography using a Chiralcel OD-H (250 mm x 4.6 mm) column and a 98 % hexanes/2 % isopropanol mobile phase at 1.75 mL min and 25 °C. The undesired (/ )-ester and desired (5)-ester were quantified using their characteristic retention times of 10.3 min and 21 min respectively during elution. 

Thin-layer chromatography (TLC) is mainly applied in micropreparative taxoids separation [2-4]. Silica gel 6OF254 preparative plates are usually applied for this purpose. The problem of taxoids separation involves not only their similar chemical structure (e.g., paclitaxel versus cephalomannine) but also, due to different coextracted compounds usually encountered in crude yew extracts (polar compounds such as phenolics and nonpolar ones such as chlorophylls and biflavones), the separation is very difficult. The common band of paclitaxel and cephalomannine was satisfactorily resolved from an extraneous fraction in isocratic elution with ethyl acetate as a polar modifier [4] and n-heptane-dichloromethane as the solvent mixture and it was of suitable purity for high-performance liquid chromatography (HPLC) quantitative determination. 

High Performance Liquid Chromatography. Tissue extracts were analyzed with a Varian model 5020 liquid chromatograph equipped with a Rheo-dyne model 7120 loop injector valve, a Tracer 970 variable wavelength detector set at 257 nm, an automated Hewlett-Packard 3385A printer-plotter system for determining retention times and peak areas, and a Waters /x Bondapak column (3.9 mm i.d. X 300 mm) for carbohydrate analysis. The buffer was eluted isocratically at 1 mL/min with a 1 4 (v/v) mixture of 0.01 M monobasic sodium phosphate (pH 4.46) and methanol. The minimum amount detectable was 10 ng. 

The determination of lovastatin and its hydroxyacid metabolite in plasma and bile can be accomplished by high performance liquid chromatography (25). Plasma samples are prepared for analysis by solid phase extraction and are analyzed using isocratic elution on a Cl 8 column. Bile samples do not require any sample dean-up prior to HPLC analysis, but do require the use of a gradient elution method to separate the compounds of interest. The HPLC assay has a limit of detection of 25 ng/mL... 

The method of characterization by high-performance liquid chromatography [HPLC] is as follows 25 mg of hemicellulose was put into 50 mL volumetric flask and pure water was added to the mark. The mixture was shaken and then filtered [first few milliliters of filtrate was discarded]. The solution was then filtered through a membrane filter of 0.2 pm cellulose nitrate. Then about 100 mL solution was injected into the HPLC system via a loop injector with a 20 mL, using an isocratic elution system with distilled water with a mobile phase, flow rate 0.8 mL/min. The detection was made using a UV detector at a wavelength of 280 nm [34]. 

Jandera P, Petranek L, and Kucerova M (1997) Characterization and prediction of retention in isocratic and gradient-elution normal-phase high-performance liquid chromatography on polar bonded stationary phases with binary and ternary solvent systems. Journal of Chromatography A 791 1-19. 

In previous papers/ we reported the effect of column temperature on resolution in reversed-phase high-performance liquid chromatography (RP-HPLC) to separate various (3-lactams (penicillins and cephalosporins) from a single sample. In this work we describe the effect of column temperature and volume fraction of an organic solvent on resolution in the isocratic elution conditions of some (3-lactam antibiotics. 

Hypoxanthine was estimated using high performance liquid chromatography on prepacked Bondapack-C18 columns (Waters Ass) eluted with an isocratic system consisting of 0.1 M KH PO, and 9% (v/v) methanol pH 5.3. 

Herve, C., Beyne, P., and Delacoux, E., 1994. Determination of thiamine and its phosphate esters in human erythrocytes by high-performance liquid chromatography with isocratic elution. Journal of Chromatography B Biomedical Sciences and Applications. 653 217-220. 

Quantitation of 1. Quantitation was performed by high performance liquid chromatography using a Hewlett Packard 1050 system. One mg of root exudates were dissolved in I mL of acetonitrile. Components of the root exudates was separated on an Alltech EPS CIS column 100 A 3p (ISO mm length x 4.5 mm internal diameter). The mobile phase was eluted at a flow rate of 2 mL/min as follows (solvent A is 2.5% acetic acid in water, solvent B is acetonitrile) 0-15 min 45% A / 55% B isocratic 15-22 min linear gradient from 55% to 100% B 22-25 min 100%B 25-26 min 100% to 55% B 26-30 min 45% A / 55% B isocratic. The peaks were monitored at X280 nm, and the volume of injection was 20 pL. Quantitation was based on a calibration curve of an external standard of purified 1. 

There is one protocol for domoic acid and analogs currently accepted, with value in commercial trading, that uses this technology to monitor marine toxins [44]. Domoic acid is extracted from a 50% mixture of methanol and water. The extract is filtered and analyzed by high-performance liquid chromatography with ultraviolet absorbance detection at 242 nm. Chromatographic separation can be performed both by isocratic or gradient elution (Table 5.5). 

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