High-Performance Liquid Chromatography (HPLC)IntroductionHigh performance liquid chromatography, also called high-pressure liquid chromatography (HPLC), separates compounds on the basis of their interaction with the solid particles of a tightly packed column and the solvent of the mobile phase. Modern HPLC, such as the one used in this laboratory, uses a nonpolar solid phase, like C18, and a polar liquid phase, generally a mixture of water and another solvent. High pressures of up to 400 bar are required to elute the analyte through the column before they pass through a diode array detector (DAD). A DAD measures the absorption spectra of the analytes to aid in their identification. HPLC is useful for compounds that cannot be vaporized or that decompose under high temperatures, and it provides a good compliment to gas chromatography for detection of compounds. Summary of MethodA Tekmar Autotract SPE workstation (Cincinnati, Ohio)* is used for solid-phase extraction. Extraction of the most common methods of analysis are described as follows: C-18, 6-cc barrel cartridges are preconditioned sequentially with 3 mL methanol, 3 mL ethyl acetate, 3 mL methanol, and 3 mL distilled water. Each 123-mL water sample is spiked with 100 µL of a surrogate standard, terbuthylazine (2.4 mg/µL), and pumped through the cartridge at a rate of 10 mL/min. Analyties are eluted with ethyl acetate and hand spiked with phenanthrene-d10. The ethyl acetate layer is transferred by hand to a clean test tube. Finally, the extract is evaporated with a Turbovap (Zymark, Palo Alto, CA)* at 45 °C under a nitrogen stream to 60 µL. There are six independent columns on the autotrace. Rinsing the probe between each sample is not necessary; however, a flush upon completion of each sample set is required. HPLC AnalysisAutomated HPLC analysis of the extracts is performed on a Hewlett Packard Model 1090 HPLC (Palo Alto, CA) with diode-array detection. The instrument parameters are as follows: analytical columns-Prodigy 5 µm ODS (3) that is 250 x 3 mm coupled to a Keystone ODS Hypersil that is 250 x 4.6 mm, 3 µm. Mobile phase is 60/35/5 pH 7 phosphate buffer/methanol/acetonitrile at a flow rate of 0.6 mL/min, analytical wave length of 210 nM, and oven temperature of 60 °C. Quantification of the analytes is achieved by dividing the peak height of the analyte by the peak height of the internal standard (2,4-D) and substituting the peak-height ratio into the respective linear regression equation. Quality assurance consists of duplicate analyses every 10 samples and agreement within 20%. Relative standard deviation for the method is ±10%. Controls are analyzed with each set at concentrations of 0.25, 0.5, 1.0, and 2.0 mg/L. The detection limit of the method is 0.2 µg/L for a sample of 123 mL. The linear range of the method is from 0.2 to 10 µg/L. Select samples are analyzed by HPLC/MS for confirmation using negative-ion electrospray. * The use of brand, trace, or firm names in this description is for identification purposes only and does not constitute endorsement by the U.S. Geological Survey. References
HPLC Available Methods
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