Hplc applications potentiometric detection.

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A new kind of detector is presented for HPLC. It uses a potentiometric sensor.

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Hplc applications potentiometric detection.

  1. 1. Potentiometric HPLC (and CE) detection Wall-jet configuration: the eluent is sprayed on the sensor. The potential between sensor and reference electrode is measured, and transformed by software to a concentration related signal. see also video: http://www.youtube.com/watch?v=LcPLiSfpgSE V
  2. 2. The heart of the system is a patented sensor. A more recent patent is pending with faster and more sensitively responding substrate and coating materials. All available HPLC examples were recorded with the old sensor, which is inferior to the new one. Potentiometric HPLC detector, beta version to be installed in selected labs
  3. 3. Alkaloids selective detection by the pot. detc., in a mix with (plant) phenolics. Green: pot. det. Blue: UV det.
  4. 4. Alkaloids with pot. det. (red) and UV det. (blue) detection limits for alkaloids are in the 10 -7 - 10 -8 M range (injected) - some 10x better than UV
  5. 5. Boldo leaf extract, crude red tracing: potentiometric detector blue tracing: UV detector
  6. 6. bromhexin 10 -5 M inj. det. lim : 2x10 -10 M, 1pg! HPLC/Potentiometry of Mucolytic drugs CN normal phase 250x4.6mm, 1mL min -1 ACN/HClO 4 1.66mM/ethanol 60:38:4 v/v 40mV One of our first applications on basic drugs: Chromatographia 57 (11-12): 757-765, 2003
  7. 7. Domperidone (a 38 μ M), enilconazole (b 12 μ M), ketoconazole (c 12 μ M), cinnarizine (d 12 μ M), and isoconazole (e 12 μ M). Waters Sunfire C18 3.5µm 100x3.5 column. Flow-rate 1.2mL min -1 , 10µL injected. From 0.1M HCOOH/20% CH 3 CN to 0.1M HCOOH/40% CH 3 CN in 30 min. 10x better detection limits than UV 10 0 2 4 6 8 0 5 10 15 20 25 time, min a b c d e 10 mV/S -1
  8. 8. 0 20 40 60 0 2 4 6 8 10 12 time, min 10 mV/S -1 a b c Aza macrocycle receptor used in the sensor: oxalic acid (a, 7 mM), malonic (b, 2 mM), and maleic (c, 4 mM). Synergi 4 µm Hydro-RP80A (Phenomenex). 0.7 mL min -1 flow-rate. Eluent: 10mM NaH 2 PO 4 pH 2.79. 10 µL injected.
  9. 9. -0,5 0,5 1,5 2,5 3,5 0 2 4 6 8 10 12 time, min C3 C4 C5 C6 C7 10 mV/S -1 1- propanesulfonic acid (C3),1- butanesulfonic, 1 - pentanesulfonic, 1 – hexanesulfonic, and 1-heptanesulfonic. 2x10 -4 M. Gravity RP 18, 1mL min -1 , gradient from10mM NaH 2 PO 4 /5% CH 3 CN (pH 2.8) to 30% CH 3 CN in 10 min. Urea receptor used in the sensor coating Time, min
  10. 10. Injection of “Zetesol” detergent (upper tracing), and a mixture of linear chain aliphatic sulfates (lower tracing). Dionex Acclaim Surfactant column (5µm 120A 4.6 x 150mm). Solvent A= 30% CH 3 CN / 10mM Na 2 HPO 4 pH 7, solvent B= CH 3 CN. From 0% B to 80% B in 30 min. 1mL min -1 , 30°C Extra receptor molecule used in the sensor coating Time, min -2 0 2 4 6 8 0 5 10 15 20 25 30 35 time, min C6 C8 C10 C12 10 mV/S -1
  11. 11. 10 mV/S -1 urine sample, male, showing naturally present dehydroepiandrosterone sulfate (DHEAS) . Dionex Acclaim Surfactant column (5µm 120A 4.6 x 150mm). Solvent A= 30% CH 3 CN / 10mM Na 2 HPO 4 pH 7, solvent B= CH 3 CN. From 0% B to 80% B in 30 min. 1mL min -1 , 30°C Receptor molecule used in coating Time, min 0 1 2 3 4 5 0 1 2 3 4 5 6 time, min
  12. 12. Chromatograms of oligonucleotides dT10, dT20, and dT30. 10 -4 M concentrations were injected. The upper and lower tracings are respectively the signals from the potentiometric detector and from the UV detector. These large, multiply charged molecules give sensitive responses, but cause peak broadening due to slow mass transfer kinetics in the above system
  13. 13. PFOS, a major environmental contaminant, is determined sensitively (lower curve). Magnification by a factor 200 (upper curve) reveals some 12 contaminants in the synthetic product. PFOS Reversed phase gradient determination of a commercial PFOS sample 20 40 0 4 8 12 time, min 10 mV/60 -1 0 0,1 0,2
  14. 14. Perfluorinated carboxylic acids The column was a MN Gravity column, eluted with a linear gradient (A solvent to B solvent in 10 min). A: 30% CH 3 CN/20mM NaH 2 PO 4 pH 2.35, B: 65% CH 3 CN/20mM NaH 2 PO 4 pH 2.35. (heptafluoro)butyric acid Ionophore used in membrane 0 2 4 6 8 10 12 14 t, min 10 - 1 C4 0 4 8 12 0 2 4 6 8 10 12 14 t, min mV/ S - 1 C3 C4 C6 C7 C8 C9 C10 N N N N C H 3 H H H N N N N C H 3 H H H N N N N C H 3 H H H
  15. 15. Quinine Clozapine Cocaine Noscapine 12 24 time, min 40mV Sample injection electrokinetically 10s at 12.5kV. Separation voltage 12.5kV. Capillary: 75 μ m i.d., 30cm length, fused silica, uncoated. Electrolyte: 50mM NaH 2 PO 4 +10% acetonitrile (pH 3.8). Only 1 example in CE is given here (Anal. Chem. 2006)
  16. 16. Conclusions for HPLC <ul><li>HPLC detection is sensitive (sub micromolar injected concentrations) and accurate. </li></ul><ul><li>It is at its best for lipophilic (logP > 2) cationic organics. Anionic organics with sulfate and sulfonic groups, dicarboxylic acids, perfluorinated carboxylic acids. </li></ul><ul><li>Also large multiply charged large molecules can be dealt with. </li></ul><ul><li>The potential for applications is very high, in industrial-, food- and biotech fields. </li></ul>

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