High performance liquid chromatography calibration curves

De Ruiter et al. [4] observed that photochemical decomposition by ultraviolet irradiation of dansyl derivatives of chlorinated phenolic compounds in methanol-water mixtures led to the formation of highly fluorescent dansyl-OH and dansyl-OH3 species. The optimal irradiation time was 5.5s. This reaction was utilised in a post-column photochemical reactor in the high performance liquid chromatography determination of highly chlorinated phenols in river water. The method calibration curve (for dansylated pentachlorophenol) was linear over three orders of magnitude. 

Recent developments in the sensitivity and resolution of NMR spectrometers permitted us to use one as a real-time detector for high-performance liquid chromatography as demonstrated in recent review articles.332,333 Recently we developed an on-line GPC/NMR system in which a 500-MHz 1H NMR spectrometer was used as a detector.334-339 By using on-line GPC/NMR the molecular weight of a polymer can be determined without a calibration curve if the polymer sample contains a known amount of end-group per polymer molecule. 

Standards a stock solution (2.5 mM) of SN-38 glucuronide (kindly supplied by Yakult Honsha Co. Ltd.) is dissolved in 5 mL of methanol. A working solution (0.5-250 iiM) for calibration curves is prepared by the serial dilution of the 2.5 mM stock solution with high-performance liquid chromatography (HPLC) elution buffer. 

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. 

Seppanen, C.M., Rahmani, M., and Csallany, A.S., Simultaneous determination of chlorophylls, pheophytins, beta-carotene, tocopherols, and tocotienols in olive and soybean oils by high-performance liquid chromatography. J. Food Sci., 68, 1644, 2003. Torsi, G., Chiavari, G., and Lippolis, M.T., Quantitation without calibration curves using HPLC with nondestructive detectors, LC-GC Inti., 5, 37, 1992. 

Fig, 4. A typical size exclusion calibration curve and chromatogram of the separation of a protein mixture. Column BIOSEP-SEC-S3000. Mobile phase pH 6.8 phosphate buffer. Detector UV abs. at 280nm. Reproduced from W.J. Lough I.W. Wainer (eds), High Performance Liquid Chromatography, 1996, first published by Blackie Academic Professional,... 

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