Analysis of oligonucleotides by electrospray ionization mass spectrometry.

Because of the high molecular weights and thermal lability of biomolecules such as nucleic acids and protein, they can be difficult to analyze by mass spectrometry. Such analyses require a "soft" ionization method that is capable of generating intact molecular ions. In addition, most mass analyzers have a limited upper mass range that is not sufficient for studying these large molecules. ESI-MS can be used to analyze molecules with a molecular weight that is larger than the mass-to-charge ratio limit of the analyzer. This unit describes how ESI allows for analysis of high-molecular-weight compounds through the generation of multiply charged ions in the gas phase. It discusses analyzer configurations, solvent selection, and gives protocols for sample preparation. For applications of ESI-MS, the unit discusses molecular weight determination and gives protocols for sequencing and for analyzing oligonucleotide modifications.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the analysis of RNase H cleavage products.

RNase H is an endonuclease which cleaves RNA at points of hybridization with DNA. However, certain ambiguities exist in terms of its specificity and location of cleavage along the RNA strand. The analysis of RNase H reaction products of an oligoribonucleotide hairpin by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS) is demonstrated. The oligoribonucleotide studied has a highly stable secondary structure which reduces the efficiency of hybridization with the chimeric oligonucleotide used to direct RNase H cleavage. By monitoring the reaction products under different conditions using MALDI/TOF-MS, the optimum variables for cleavage of this highly stable hairpin structure can be determined.

HPLC-Electrochemical detection with graphite-poly (tetrafluoroethylene) electrode Determination of the fungicides thiram and disulfiram.

The suitability of composite graphite-poly(tetrafluoroethylene) (Teflon) electrodes as amperometric indicator electrodes in HPLC detection is demonstrated. The determination of the fungicides thiram and disulfiram in the presence of ziram has been chosen as an analytical problem. The optimization of working conditions, such as the choice of the organic solvent used in the mobile phase as well as its percentage, the potential applied to the composite electrode, and the time elapsed between mixing the carbamates and the injection, has been accomplished by using the wall-jet flow-cell configuration. The effect of the acetonitrile percentage used in the mobile phase on the retention of thiram, disulfiram, ziram and phenol was evaluated. Resolution up to the baseline can be achieved with 45% acetonitrile. The sensitivity of the determination of thiram and disulfiram in the presence of a constant concentration of ziram is slightly better when using a wall-jet cell; however, the background current is higher, as well as the baseline noise and the time necessary to achieve stabilization of the baseline before the injection. Lower limits of detection for both fungicides, as well as a better repeatability, were obtained when using a thin-layer flow cell configuration. As an application, the determination of thiram in spiked apple samples, at a level of 0.5 mg thiram kg(-1) apple, has been carried out with a mean recovery of 97 +/- 3% for a significance level of 0.05.

Differential pulse polarographic study of the hydrolysis of endosulfan and endosulfan sulphate in emulsified medium. Application to the determination of binary mixtures of organochlorine pesticides.

Hydrolysis reactions of endosulfan and endosulfan sulphate in the emulsified medium formed with ethyl acetate and a mixture of the two surfactants Hyamine 2389 and Triton X-405 are studied by differential pulse polarography. Besides the heptachlor-endosulfan sulphate pair, whose peak potentials are sufficiently different at pH 8.0 to allow their simultaneous determination, the organochlorine pesticide binary mixtures endosulfan-endosulfan sulphate, dieldrin-endosulfan and dieldrin-endosulfan sulphate can be determined based on their hydrolysis reactions in basic medium and on their different reaction rates. The endosulfan-endosulfan sulphate pair can be determined by allowing the mixture to hydrolyse at pH 11.0 and measuring the endosulfandiol peak for the determination of endosulfan. The analysis of the mixture dieldrin-endosulfan is based on endosulfan hydrolysis at pH 12.0 in which dieldrin is not hydrolysed. Dieldrin and endosulfan sulphate can also be determined simultaneously in a 0.5M sodium hydroxide medium. When determining one pesticide in binary mixtures containing a 5.0 x 10(-6)M concentration of the other pesticide, the lower limits of the calibrations obtained were: endosulfan-endosulfan sulphate mixture, 4.0 x 10(-6) and 1.0 x 10(-6)M respectively; heptachlor-endosulfan sulphate mixture, 2.0 x 10(-6)M for endosulfan sulphate; all other cases, 3.0 x 10(-6)M.

Transient changes of mobile phase in the high-performance liquid chromatographic separation of priority pollutant phenols.

A study of the effect of cetyltrimethylammonium bromide on the separation of the eleven priority pollutant phenols is presented. Transient changes in a CTAB mobile phase produced by a sodium laurylsulphate solution plug permit the elution of hydrophobic pentachlorophenol.