New Tool: Potentiometric HPLC Detector

Potentiometric detection of organic substances in HPLC: an emerging technique.

Luc Nagels Antwerp University Potentiometry has many faces Like these ceramics from Cracau, potentiometry has many faces

Poland, Olsztyn, September 2003 Old: cationic: metals, drugs and amines 2- - - - - anionic: S , SCN , NO3 , NO2 and ClO4 , org.acids Undiscovered: charged organic biochemicals, biogenic amines, oligonucleotides, proteins, (hydrophilic and/or expensive) Lecture: drugs and amines .. how does it work .. biomolecules .. CE

New tool:

potentiometry in LC + CE: embryonal form potentiometric HPLC detector Nr. of publications on potentiometric detection in LC + CE (5 year periods - mostly inorganic ions): 70 conductive poly- and oligomers, ceramics, liquid membranes

determination of organic cationic analytes, HPLC Potentiometric sensors for LC or CE

analytical signal, to substrate electrode data station PVC/plasticizer reference rubber phase coating electrode

neutral ionophore lipophilic anion like p-Cl tetraφ borate membrane coating on and buffer countercation substrate electrode

LC or CE outlet

1 Potentiometric det. of linear aliphatic amines potentiometric detection of beta-adrenoceptors in RP

2 1 clenbuterol 2 1 2 practolol 3 4 5 3 oxprenolol 3 4 5 6 4 alprenolol 6 5 carazolol 1 4 mV 6 bevantolol 7,8 unknowns

212 Time, min 6 -6 1 Methylamine 10 M injections, 20µL … PVC/DOS/TCPB 32:66:2 wt% 5 6 Hexylamine X-Terra RP18, 50mmx3mm, Alltech universal cation exchange 2 4 5×10-5 M acetonitrile/20mM H PO , 25:75 1 3 3 4 4.6 mm ID, 5mMHNO3 in 5% inj., 50µL acetonitrile, 1mL min-1, Journal of Chromatography A, 973, 85-96, 2002

Analytica Chimica Acta 440 (2) 89-98, 2001.

lysosomotropic alkanolamines detection limits (molar conc., 20 L injected) µ 1 analyte UV 230nm Borate OctylCD Calix mol/L mol/L mol/L Alltech universal cation exch. Clenbuterol 2.6 x 10-7 6.7 x 10-7 1.1 x 10-7 1.7 x 10-6 -6 -7 -8 -5 40 mM H PO /acetonitrile 85:15 Mabuterol 2.9 x 10 2.2 x 10 5.6 x 10 3.4 x 10 35 mV 3 4 -7 -5 -6 -5 Cimaterol 3.0 x 10 2.5 x 10 4.8 x 10 3.6 x 10 Inj. conc.: 2x10-4M, 20 microL Carbuterol 1.7 x 10-7 8.5 x 10-5 7.7 x 10-5 2.4 x 10-4 Bufuralol 3.5 x 10-7 2.8 x 10-7 2.2 x 10-8 1.5 x 10-6 2 -7 -5 -5 -4 Practolol 1.7 x 10 5.4 x 10 6.2 x 10 1.9 x 10 3 1: aminoethanol Alprenolol 2.2 x 10-6 6.7 x 10-7 3.6 x 10-7 3.0 x 10-7 2: N-methylaminoethanol Oxprenolol 1.0 x 10-6 1.7 x 10-6 7.7 x 10-7 5.8 x 10-7 5 6 p-Oxprenolol 0.7 x 10-6 1.6 x 10-6 7.5 x 10-7 3.5 x 10-7 4 3: N-ethylaminoethanol Bisoprolol 3.5 x 10-7 1.3 x 10-5 3.7 x 10-6 2.7 x 10-6 Celiprolol 1.6 x 10-7 1.7 x 10-5 1.6 x 10-5 6.8 x 10-5 4: N,N-dimethylaminoethanol Bevantolol 1.3 x 10-6 2.2 x 10-6 1.0 x 10-6 1.6 x 10-6 5: N,N -dimethylaminoisopropanol Tolamolol 1.3 x 10-6 1.4 x 10-5 1.3 x 10-6 2.0 x 10-5 Propranolol 2.7 x 10-8 2.1 x 10-7 1.0 x 10-7 5.3 x 10-7 6: N,N-diethylaminoethanol -7 -5 -5 -4 Nadolol 2.3 x 10 5.9 x 10 2.1 x 10 1.0 x 10 0 time, min 2 Tertatolol 1.2 x 10-6 3.5 x 10-7 1.2 x 10-7 1.8 x 10-6 Analytica Chimica Acta, 472, 11-26, 2002. Carazolol 4.4 x 10-7 6.0 x 10-4 2.0 x 10-5 2.1 x 10-4

detection limits (molar conc., 20µL injected)

Solute LogP UV210nm Calix mol/L mol/L

1. Aminoethanol -1.82 8.3 x 10-5 5.0 x 10-7 2. N-Methylaminoethanol -1.27 5.8 x 10-5 1.1 x 10-8 3. N-Ethylaminoethanol -0.86 2.1 x 10-4 5.2 x 10-7 potentiometry: harnessed knight or nice lady? 4. N-tert-Butylaminoethanol +0.05 4.2 x 10-5 2.5 x 10-5 5. N.N-Dimethylaminoethanol -0.58 9.7 x 10-5 1.4 x 10-6 - new materials and receptors please 6. N.N-Diethylaminoethanol +0.27 9.0 x 10-5 1.3 x 10-6 7. N.N-Dimethylaminoisopropanol -0.21 1.7 x 10-4 6.7 x 10-6 - “plagued by fundamental errors and lack of 8. N-Butyldiethanolamine -0.10 1.2 x 10-4 4.1 x 10-7 conceptualization”: K.L. Cheng, Microchem. J., 2002, 72, 269 9. N-tert-Butyldiethanolamine +0.01 4.5 x 10-3 2.0 x 10-6 10. N-Phenyldiethanolamine +0.43 4.0 x 10-7 6.3 x 10-6 11. N-(2-Hydroxyethyl)-ethylendiamine -2.07 2.7 x 10-4 2.0 x 10-5

4 6

2 liquid membrane phase + + + How to predict the sensitivity of non-selective membranes for analytes?

E QSAR (Multiple correlation model), 8 physicochemical parameters, 18 betablockers

Eluent phase logDL = - 7.0190 - 0.7967 logP + 0.0815P r2 = 0.81 n=18 +

high extraction tendency → low DL low polarizability ∆G = ∆G -(∆G + ∆G + ∆G ) high lipophilicity ion-ion interactions tr hydr solv ion complex detection limit membrane solubility

logDL = a - blogP + cPvol Analytica Chimica Acta, 472, 11-26, 2002. Intermolecular interactions → “non-faradaic” potentiometry

Nagels, L. J.; Bazylak, G.; Zielinska, D. Electroanalysis 2003, 15, 533-38.

CH3 Br effect of logP and B-solvent on N sensitivity NH 2 40mV Br

CH 3 -9 Br Mucolytic drugs, potentiometric N 10 M bromhexin injected

NH detection (“TOPA” ionophore) bromhexin2 Br bromhexin 10-5M 1mV 40mV OH injected Br N CN Uptishere, normal phase H -1 NH 250x4.6mm, 1mL min 2 Br ACN/HClO4 1.66mM/ethanol ambroxol 60:38:4 v/v pH 2.45 clenbuterol 0 min 4 Normal-phase, CN det. lim.bromhexin: 2x10-10M, 1pg!

0 4 min

application: human saliva spiked Researchers do not walk like sheep in with clenbuterol, no cleanup, the Tatra mountains.. standard RP OH H Can we do large biomolecules? Can we Cl N * use solid-state electrodes? Is miniaturization possible? H2N Cl 10-4M

10-5M 10-6M

0 3 min

3 UMP AMP nucleotides UV X-terra C18 (3x50mm), ion-pair Oligonucleotides, FIA 4x10-5M inj in 10µL 25-mer CAGACAATAGTAAATGAGAATCAGG

4mV -5 10 M in 1mM H3PO4 potentiometric 0.3mL/min, 10 µL injection UV Potentiometry 0.71mV UDP UTP ADP ATP

0 60 time, s 0 5 time, min 10

+ Oligonucleotides d(T)12-18 5‘OH Na salt V

X-Terra RP18 3x50mm 10mM TEAE, 0.3mL min-1, 8 to 10% AcN Substrate electrode Potentiometry Interface A 2mV UV Liquid membrane phase

Interface b E Eluent phase

45 6 7 8 9 10 11 12 13 14 15 16 17 18-mer 0 9 volume, mL 18 Do we need faradaic processes (ion-electron conversion) at interface A? Lack of knowledge on ionic-electronic circuitry unpublished results

CE of linear chain aliphatic amines CE with potentiometric detection, straight chain Potentiometric detection - polymeric carboxylic acids (C2 to C10) electrode, 0.25mm C6 C5 75 um, 60cm, 15kV, 50mM NaH2PO4 9 7

10 8 6 5 4 3 2 C4 10mV 20mV 5mM Hepes pH 7.6, 22kV, 66cm, 75µm ID, C3 injected concentrations: 10-4M C2 C1 10-3 M inj 0 4 8 12 time, min 10-4 M inj C2 C3 C4 C5 C6

0 10 time, min 20

4 Potentiometric detection for HPLC and CE works. It can be made extremely sensitive for organic ionizable substances. New classes of compounds can be studied, especially bio- organics. New electrode materials are needed to handle hydrophilic organic substances, and new receptors. Thanks to Grzegorz Bazylak, Danuta Zielinska, Jozef Everaert, Lily van Roy, Ying Bao, Hugo Bohets, Bert Vissers, David Dehous,Tom Wuyts, Justyna Sekula Thanks to the Belgian-Polish bilateral project