Conductivity detection of polymerase chain reaction products separated by micro-reversed-phase liquid chromatography.
In this paper, we describe the application of micro-reversed-phase high-performance liquid chromatography (mu-RP-HPLC) for the separation and/or purification of polymerase chain reaction (PCR) products with detection accomplished using a miniaturized conductivity detector. The conductivity detector used two Pt wires and a bipolar waveform applied to the electrode pair from which the conductivity of the bulk solution could be measured. In the mobile phase used for the mu-RP-HPLC separation of the PCR product, the mass detection limit for herring sperm DNA using conductivity was found to be 11 ng. Efficient separation of the PCR amplicon from the other reagents present in the PCR cocktail was achieved in less than 4 min with a capacity factor of 2.5 and separation efficiency of 9.1 x 10(3) plates. The separation was carried out using reversed-phase ion-pair chromatography with a triethylammonium acetate ion-pairing agent.
McWhorter
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Department of Chemistry, Louisiana State University, Baton Rouge 70803-1804, USA.
Soper
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SA
D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004553:Electric Conductivity; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity