Determination of dissolved thiols using solid-phase extraction and liquid chromatographic determination of fluorescently derivatized thiolic compounds.
A method employing solid-phase extraction coupled with HPLC separation of thiol-monobromobimane (mBBr) derivatives was developed and optimized to quantify dissolved thiols at concentrations as low as 0.1 nM for glutathione (GSH) and gamma-glutamylcysteine (gammaEC) in natural waters. The reducing reagent, tri-n-butylphosphine (TBP), is needed for complete derivatization. At the optimal addition of TBP ([TBP]/[mBBr] = -0.4-1.6), no interference from copper was observed. The thiol fluorescence signal was totally suppressed if the mole ratio of TBP to mBBr was 2.6 or greater. Consistent recovery of thiols standards in a NaCl solution (0.5 M) was obtained using the Waters HLB reversed-phase resin, and blank levels of GSH and gammaEC were extremely low (less than 0.03 nM). The detection limits for GSH, gammaEC and phytochelatin-2 (PC-2) were 0.03, 0.03, and 0.06 nM, respectively.
Tang
Degui
D
Environmental Chemistry and Technology Program, University of Wisconsin-Madison, 660 North Park Street, Madison, WI 53706-1484, USA. deguitang@facstaff.wisc.edu
Shafer
Martin M
MM
Vang
Kou
K
Karner
Dawn A
DA
Armstrong
David E
DE
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D007700:Kinetics; D012015:Reference Standards; D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds / Q000032:analysis*; D013696:Temperature
D005456:Fluorescent Dyes; D013438:Sulfhydryl Compounds