Fig. 1 DECADE Electrochemical Detector equipped with a build-in electrically-actuated valve (Valco). Column Spherisorb S3 ODS2, 100 x 4.6 mm I.D., particle size 3 µm (Waters, USA) Flow rate 1 mL/min Eluant 50 mM phosphoric acid, 50 mM citric acid, 100mg/l octane sulphonic acid, 40 mg/l EDTA, 2 mM KCl, 5% MeOH, pH 3.0 Sample I 0.1 µM norepinephrine standard in 10 mmol/L perchloric acid (PCA). Sample II 10 nM epinephrine standard in Ringer solution (Ringer: 147 mM NaCl, 3 mM KCl, 1.2 mM CaCl 2 and 1.2 mM MgCl 2 ). This salt matrix was chosen to induces a large front peak / baseline disturbance. V sample inj. 20 µL Detector DECADE with build-in electrically-actuated Valco valve (Antec Leyden, The Netherlands) T oven 30 o C Cell VT-03 wall-jet flow cell with 1.9 mm glassy carbon electrode and in-situ Ag/AgCl reference electrode (ISAAC), spacer thickness 50 µm, V cell ~140 nL (Antec Leyden, The Netherlands) E cell Sample I : 650 mV, 750 mV, 850 mV and 950 mV (vs. Ag/AgCl in 2 mM KCl), sample II: 650 mV (vs. Ag/AgCl in 2 mM KCl) Sensitivity Sample I : 10 nA/V, sample II: 50 pA/V (1 V FS) The removal of the solvent front and contaminants in HPLC-ECD by means of a post-column valve H.J. Brouwer, N.J. Reinhoud and C.F.M. van Valkenburg Antec Leyden B.V., Industrieweg 12, 2382 NV Zoeterwoude, The Netherlands Set-up Introduction Experimental conditions Hig ve h Performance Liquid Chromatography combined with electrochemical detection (HPLC-ECD) has evolved to a rsatile and powerful analytical technique used for the (trace) analysi of a wide range of compounds such as biogenic amines, phenols, vitamins, inorganic ions, DNA adducts, amino acids and carbohydrates [1-4]. A consequence of the extreme sensitivity of ECD is that high concentrations of substances eluting in the unretained ‘system peak’ may have an impact on the robustness and reproducibility of a method. An automated switching valve fitted between the column and the electrochemical flow cell can be a useful accessory to reduce fouling of the WE. It offers the possibility to selectively divert the solvent front/electrochemical contaminants to waste, preventing exposure of these compounds to the WE [5]. In this poster experimental data are presented which demonstrate the stabilisation of the flow cell in less than 1 s after (post-column) valve switching at high detector sensitivities (nA and pA range). An example of the removal of the solvent front during the HPLC-ECD analysis of dialysate containing 10 nM ephinephrine is given. s Stabilisation behaviour of the electrochemical flow cell I. Analysis of 2 pmol norephinephrine Fig. 2A Analysis of 2 pmol norepinephrine in 10 mM PCA (removal of the PCA front by means of the post-column valve). Chromatograms: (A) Normal run, valve not used (B) flow to cell restored at t = 2.06 min (C) t = 2.30 min (D) t = 2.50 min and (E) t = 4.00 min. Settings: E cell = 750 mV (vs. Ag/AgCl in 2 mM KCl), range 10 nA/V (1 V full scale). PCA 1 nA To waste To cell E D C B A Norepinephrine (2 pmol) HO HO NH 2 OH 0.0 0.5 1.0 1.5 2.0 2.5 overshoot E cell = 650 mV E cell = 750 mV E cell = 850 mV E cell = 950 mV to waste to cell Normalised cell current Time (min) -0.4 0.0 0.4 0.8 1.2 1.6 2.8 5.9 to cell to waste Cell current (nA) B C D E Stabilisation time (s) Fig. 3 Recovery of cell current after valve switching at different cell potentials: 650 mV, 750 mV, 850 mV and 950 mV (vs. Ag/AgCl in 2 mM KCl). The flow to cell was blocked at t = 5 s and restored again at t = 2.06 min. Fig. 2B Recovery of cell current for case (B) flow to cell restored at t = 2.06 min (C) t = 2.30 min (D) t = 2.50 min and (E) t = 4.00 min (all slopes of the chromatograms B to D of figure 2A superimposed). Conclusions References Fig. 4 Analysis of 200 fmol epinephrine in ringer (A) without removal of the ringer front, (B) with removal of the ringer front, flow to cell restored at t = 3.21 min. Settings: E cell = 650 mV (vs. Ag/AgCl in 2 mM KCl), range 50 pA/V (1 V full scale). II. Analysis of 200 fmol ephineprine 0 1 2 3 4 Post-column valve to waste to waste OVERLOAD OVERLOAD Normal run, no valve used Epinephrine (200 fmol) to cell 10 pA Time (min) HO HO N OH CH 3 H [1] D.C. Johnson, S.G. Weber, A.M. Bond, R.M. Wightman, R.E. Shoup and I.S. Krull, Anal. Chim. Acta, 180 (1986) 187-25 [2] H. Parvez, M. Bastart-Malsot, S. Parvez, T. Nagatsu, and G. Carpentier, Electro analytical measurement in Medicine and Chemistry, VNU science press, Utrecht, Netherlands, 1987 [3] G. Horvai and E. Pungor, Crit. Rev. Anal Chem., 21 (1989) 1-28 [4] W.R. LaCourse, Pulsed electrochemical detection in HPLC, Wiley, New York, 1997 [5] J. Lagendijk, J.B. Ubbink and W.J. Hayward Vermaak, J.of Lipid Research, 37 (1996) 67-75 The presented data illustrate the fast current stabilisation of the VT03 flow cell after post-column valve switching at high detector sensitivities (pA range). Allowing the removal of contaminants/solvent front up to seconds prior to elution of the analyte of interest during HPLC-ECD trace analysis. Especially the analysis of samples with “dirty” matrices, where the analyte(s) of interest does not have a large contribution to WE fouling could benefit from a post-column valve. The utilisation of an automated post-column valve in a HPLC-ECD system is fairly simple and optimum performance can easily be achieved by using low dead volume connections and by proper adjustment of the backpressure on both flow channels (to reduce peak broadening and baseline instabilities, respectively). www.antecleyden.com mailbox@antecleyden.com +31 (71) 581 3333