Potentiometric sensing of organics for HPLC and CE: Ready ... filePotentiometric sensing of organics...
Transcript of Potentiometric sensing of organics for HPLC and CE: Ready ... filePotentiometric sensing of organics...
Potentiometric sensing of organics for HPLC and CE: Ready for takeoff!
Luc NagelsAntwerp University
Hergé, (Belgium)
Potentiometric sensors for:HPLC detection, CE detection, dissolution testing
conductive poly- and oligomers, ceramics, liquid membranes
Potentiometry, the "sleeping lion"
Hunting: metal ions, cationic drugs and amines, halogen ions, S2-, SCN-, NO3
-, NO2- and ClO4
-, organic acids, …
Not hunting: biomolecules, multiply charged biomolecules
Why not? too hydrophilic, too expensive, too complex
Pure and Applied Chemistry, to be published 2004
Potentiometry/HPLC or CE or microchip hyphenation
Application in HPLC: 50 older references
Application in CE and microchip: 20 recent references
Electroanalysis, 15 (5-6): 533-538, 2003.
Potentiometric sensors for LC and CE
data station
reference electrode
coating on electronic conductor
LC or CE outlet
plastic wire
Electronic conductor
Ionically conducting membrane
E
Receptor molecules +
Electronic conductor substrate electrode (or internal solution).
membrane surfaces!
EAnalyte solution
A potentiometric sensor has one or two important surfaces
E depends on analyte concentration
+
E
++++
++++
Substrate electrode
Liquid membrane phase (polymer + plasticizer + ionophore)
analyte anion counter cation
is the analyte ion extracted or adsorbed?
1 Tartaric acid2 Malonic acid3 Malic acid4 Lactic acid5 Citric acid6 Fumaric acid7 Succinic acid
2 mV1
2
3
4
5
67
Column: RPC8, 4.6mm i.d.Eluent: 1 mM H3PO4, 1mL/min
pg detection limits for malonic0 10Time, min
++++
+
lipophilic tail
polar head
“embedded” charge, MTDDACl (methyltridodecylammoniumchloride)
NH
NH
O
NNH2
C18H37
NH
NHO
N
NH
NHO
C18H37
NH
NHO
C18H37C18H37
NH
NHO
N
NH
NHO
NH
NHO
NH
NH
O
NC18H37 N
HNH
O
C18H37
ligand 1 ligand 2
ligand 3 ligand 4
RRN
NH
N
NH
N
NH
NH
NRR
ligand 6 R = H, R1 = C10H21ligand 8 R = R1 = (CH2)3NHCONHC18H37ligand 9 R = R1 = CH2CONHC2H4N(C2H4NHCONHC18H37)2
R R1
RR1
R
N
N
N
N
N
N
R
ligand 7 R = C10H21
ligand 5 R = C8H17
R
RR
R
NN
N
N
N
N
R
R
ligand 10 R = (CH2)4OCONHC18H37
R N
N
N
N
N
NCH3
CH3
CH3
CH3
CH3
0.2% MTDDACl (embedded type), and 1% ligand (head/tail type)
Lichrosphere 100-5 RP8 column (Merck), 125x4mm, eluens 1mM H3PO4 at 0.5 mL min-1 flow-rate. 10 µL injection of a mixture of tartaric- (1, 10-5M ),malonic- (2, 2x10-7M), malic- (3, 4x10-6M), lactic- (4, 10-4M), citric-(5, 10-
5M), fumaric- (6, 2x10-5M), succinic acid (7, 2x10-5M).
5
25 50concentration, Mì
10
peak height,mV
injected micromolar concentration
Calibration curves obtained with the HPLC potentiometric sensor. Upper curve: malonic acid, middle curve: tartaric acid, lower curve: citric acid.
+
E
Substrate electrode
Liquid membrane phase
Eluent phase
++
+
“Embedded” charge type
versus head/tail type
malonic acid
docking, molecular modeling, gasphase
modeling interface behavior
+++
- -
UMP AMP
UDP UTP ADP ATP
X-terra C18 (3x50mm), ion-pair
4x10-5M inj in 10µL
0.71mV
0
nucleotides
UV
potentiometric
0 5 time, min 10
Oligonucleotides, FIA
25-mer CAGACAATAGTAAATGAGAATCAGG
0 60 time, s
UV Potentiometry
4mV 10-5M in 1mM H3PO4
0.3mL/min, 10 µL injection
0 9 volume, mL 18
45 6 7 8 9 10 11 12 13 14 15 16 17 18-mer
Oligonucleotides d(T)12-18 5‘OH Na+ salt
X-Terra RP18 3x50mm
10mM TEAE, 0.3mL min-1, 8 to 10% AcN
2mV
Potentiometry
UV
unpublished results
-
E
Substrate electrode
Liquid membrane phase
Eluent phase
p-chloro tetraphenylborate
+-
++
sensing organic cationic substances
Potentiometric det. of linear aliphatic amines
6543
2
1 4 mV
123 4
5
6
123
4
5
61 Methylamine
…6 Hexylamine
Alltech universal cation exchange 4.6 mm ID, 5mMHNO3 in 5% acetonitrile, 1mL min-1,
2 12Time, min
5×10-5 M inj., 50µL
Analytica Chimica Acta 440 (2) 89-98, 2001.
1 clenbuterol 2 practolol 3 oxprenolol 4 alprenolol 5 carazolol 6 bevantolol 7,8 unknowns
potentiometric detection of beta-adrenoceptors in RP
10-6 M injections, 20µL
PVC/DOS/TCPB 32:66:2 wt%
X-Terra RP18, 50mmx3mm,acetonitrile/20mM H3PO4, 25:75
Journal of Chromatography A, 973, 85-96, 2002
detection limits (molar conc., 20µL injected)analyte UV 230nm Borate OctylCD Calix
mol/L mol/L mol/L
ClenbuterolMabuterolCimaterolCarbuterolBufuralolPractololAlprenololOxprenololp-OxprenololBisoprololCeliprololBevantololTolamololPropranololNadololTertatololCarazolol
2.6 x 10-7
2.9 x 10-6
3.0 x 10-7
1.7 x 10-7
3.5 x 10-7
1.7 x 10-7
2.2 x 10-6
1.0 x 10-6
0.7 x 10-6
3.5 x 10-7
1.6 x 10-7
1.3 x 10-6
1.3 x 10-6
2.7 x 10-8
2.3 x 10-7
1.2 x 10-6
4.4 x 10-7
6.7 x 10-7
2.2 x 10-7
2.5 x 10-5
8.5 x 10-5
2.8 x 10-7
5.4 x 10-5
6.7 x 10-7
1.7 x 10-6
1.6 x 10-6
1.3 x 10-5
1.7 x 10-5
2.2 x 10-6
1.4 x 10-5
2.1 x 10-7
5.9 x 10-5
3.5 x 10-7
6.0 x 10-4
1.1 x 10-7
5.6 x 10-8
4.8 x 10-6
7.7 x 10-5
2.2 x 10-8
6.2 x 10-5
3.6 x 10-7
7.7 x 10-7
7.5 x 10-7
3.7 x 10-6
1.6 x 10-5
1.0 x 10-6
1.3 x 10-6
1.0 x 10-7
2.1 x 10-5
1.2 x 10-7
2.0 x 10-5
1.7 x 10-6
3.4 x 10-5
3.6 x 10-5
2.4 x 10-4
1.5 x 10-6
1.9 x 10-4
3.0 x 10-7
5.8 x 10-7
3.5 x 10-7
2.7 x 10-6
6.8 x 10-5
1.6 x 10-6
2.0 x 10-5
5.3 x 10-7
1.0 x 10-4
1.8 x 10-6
2.1 x 10-4
35 mV
0 2time, min
lysosomotropic alkanolamines
Alltech universal cation exch.
40 mM H3PO4/acetonitrile 85:15
Inj. conc.: 2x10-4M, 20 microL
654
32
1
1: aminoethanol
2: N-methyl- aminoethanol
3: N-ethyl- aminoethanol
4: N,N-dimethyl- aminoethanol
5: N,N -dimethylaminoisopropanol
6: N,N-diethyl- aminoethanol
Analytica Chimica Acta, 472, 11-26, 2002.
E
liquid membrane phase
++
+
Eluent phase+
high extraction/adsorption tendency → low DL
∆Gtr = ∆Ghydr - (∆Gsolv + ∆Gion + ∆Gcomplex)
logDL = a - blogP + cPvol
Intermolecular interactions → “non-faradaic” potentiometry
Nagels, L. J.; Bazylak, G.; Zielinska, D. Electroanalysis 2003, 15, 533-38.
40mV
bromhexin
ambroxol
clenbuterol
0 4 min
bromhexin 10-5M inj.
det. lim: 2x10-10 M, 1pg!
HPLC/Potentiometry of Mucolytic drugsCN normal phase 250x4.6mm, 1mL min-1 ACN/HClO4 1.66mM/ethanol 60:38:4 v/v
40mV
Br
Br
NH2
NH
OH
Br
Br
NH2
N
CH3
Chromatographia 57 (11-12): 757-765, 2003
0 min 4
10-9M bromhexin injected
1mV
Br
Br
NH2
N
CH3
Normal-phase, CN det. lim.bromhexin: 2x10-10M, 1pg!
Liquid membrane phase (ionic conductivity)
Substrate electrode(electronic conductivity)
Interface A
V
E
Interface A gives interfacial tensions depending on eluent composition. Little is known on substrate/membrane adhesion chemistry
CE of linear chain aliphatic amines
Potentiometric detection - polymericelectrode, 0.25mm
75 um, 60cm, 15kV, 50mM NaH2PO4
C6
C5
C4
C3
C110-3 M inj
10-4 M inj
10mV
C2
C2 C3 C4 C5 C6
0 10 time, min 20
Time [min]17127 22 27 32 37 42
40 mV
Cocaine
HeroinAmine
Electropherogram of aliphatic amines (1-6), heroin (7), and cocaine (8). Concentrations injected: 10-3M (amines), 10-4M (drugs). Cyclodextrin was added to the eluent.
Quinine
Clozapine
Cocaine
Noscapine
40mV
12 24 time, minSample injection electrokinetically 10s at 12.5kV. Separation voltage 12.5kV. Capillary: 75 µm i.d., 30cm length, fused silica, uncoated. Electrolyte: 50mM NaH2PO4 +10% acetonitrile (pH 3.8).
Cocainey = 73.29x + 380.8
R2 = 0.9993
20406080
100120140160180
-5.5-5-4.5-4-3.5-3
log [c]
mV
It did not always go that easy..
Electropherogram
hexanoic- (6), pentanoic- (5), butyric- (4), propionic- (3), acetic-(2) and formic acids
5x10-4M in 5mM HEPES (pH 7.6). Electrokinetic inj. 10s 5kV
75µm i.d., 60 cm, fused silica, uncoated, +20kV
Separation voltage: +20kV Electrolyte: 5mM HEPES pH 7.6
Anal. Chim. Acta 401, 21-27, 1999
CO
PPy electrode
Also terrible...
Same acids in HEPES containing 0.5mM TTAB (tetradecyltrimethylammoniumbromide)
A: conducting trimer electrode, phenylene vinylene type, plus polycarbonate
B: PPy electrode
-20kV (negative polarity)
Potentiometric detection for HPLC and CE can be made extremely sensitive for organic ionizable substances.
New classes of compounds can be studied, especially bio-organics.
More understanding of surface chemistry is needed
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