Determination of phosphorus and carbon in phosphorylated deoxynucleotides via particle beam/hollow cathode glow discharge optical emission spectroscopy (PB/HC-OES)

Abstract

A new approach to the qualitative and quantitative determination of nucleic acids is presented. Particle beam hollow cathode optical emission spectroscopy (PB/HC-OES) has been applied to determinations of five nucleotides (AMP, ADP, dATP, dCTP, and dGTP) by monitoring P I 219.9 nm and C I 193.0 nm optical emission. A high level of correlation exists between the phosphorus emission response and the degree of phosphorylation, demonstrating the technique's ability to produce vital qualitative and quantitative information for specific nucleotides, with detection limits on the single-nanogram level for phosphorus (9 ng) and picogram level for carbon (20 pg), respectively, with RSD o12% for triplicate injections over a concentration range of 10 À4 -10 1 mg mL À1 . The stoichiometric response suggests that it is possible to identify the various nucleotides by the amount of phosphate present within the nucleotide. Empirical formula calculations on P I and C I emission response ratios demonstrate the potential of the technique as an element specific detector for liquid chromatography (LC).

Simultaneous multielement detection in particle beam/hollow cathode-optical emission spectroscopy

“…The optimum temperatures observed here are appreciably higher than previous works (∼ 200°C) [10,11,16,19,20]. This difference can be attributed to the source block geometry changes and the size and amount of boron nitride used in the new design of the thermoblock.…”

“…While it is easy to suggest that higher temperatures would be desirable, temperatures could not exceed 500°C due to melting of the Teflon O-ring used in the top of the HC source block, causing loss of vacuum integrity. In addition, temperatures exceeding 500°C would also be expected to yield a decrease in analyte response for organic species due to pyrolysis [10,11,16,19,20]. Further refinement may be required to efficiently detect inorganic and organic (metal and nonmetal) species simultaneously.…”

“…It has been previously shown that vaporization from the HC walls is the primary means of generating gas-phase species in the PB/HC-OES system [16,19]. Optimal HC temperatures have ranged from 200-300°C, with higher temperatures leading to pyrolysis of carbonaceous samples [10,11,16,19,20]. Given the difference in the placement of the heaters and thermocouples in the new design, it was imperative to characterize the role of "measured" temperature on the analyte responses.…”

“…The HC glow discharge design was altered slightly from the one employed in previous works [10][11][12][13][15][16][17][18][19]21,22]. The HC was machined from a bulk copper rod with an inner diameter of 3.5 mm Fig.…”

“…Techniques supplying both molecular and atomic information would be very powerful contributors in the field of metallomics. This laboratory has previously developed a liquid chromatography-particle beam/hollow cathode-optical emission spectrometer (LC-PB/HC-OES) that can analyze inorganic and/or organic samples, providing empirical formulas based on element ratios (including C, H, N, O, S, and metals), with detection limits on the single ng mL − 1 level and below [10][11][12][13]. The PB/HC-OES system combines the excitation properties of a glow discharge (GD) source with the solvent removal capabilities of the particle beam (PB) to allow dry analyte particles to reach the hollow cathode (HC) GD region [7].…”