Hplc applications potentiometric detection.
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Transcript of Hplc applications potentiometric detection.
Potentiometric HPLC (and CE) detection
V
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
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
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45UV 200nm Pot trans
Ergocornine 4.2x10-4 M
cocaine 1.5x10-4 M
time, min
POT, mVUV, AU200nm
Solanidine
6x10-5 M
Metergoline
2.5 10-4 M
Column: MN-Nucleodur 100-5 CN-RP 125/4mmInj: 20µLFlow: 1mL/minEluent: A=5mM H3PO4
Eluent: B=30% CH3CN / 5mM H3PO4
Gradient: 25%B to 100%B in 30minElectrode 126: PVC 3mm diam comp2 1mm diamMembrane: PVC DOS TCPB 2x 3.5µL
p-coumaric acid
1.5x10-4 M methylester p-coumaric acid
1.5x10-4 M
ethylester p-coumaar
acid 1.5x10-4 M
Ergotamine
1.5x10-4 M
impurity ergocornine
Alkaloids selective detection by the pot. detc., in a mix with (plant) phenolics. Green: pot. det. Blue: UV det.
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80UV 200nm POT
Ergocornin
e 7 10-5 Mcocaine 2.5 10-5 M
time, min
POT, mVUV, AU200nm
Solanidin
e 10-5 M
Metergoline
1.7 10-5 M
Column: MN-Nucleodur 100-5 CN-RP 125/4mmInj: 20µLFlow: 1mL/minEluent: A=5mM H3PO4
Eluent: B=30% CH3CN / 5mM H3PO4
Gradient: 25%B to 100%B in 30minElectrode 123: PVC 3mm diam comp2 1mm diamMembrane: PVC DOS TCPB 2x 3.5µL
Alkaloids with pot. det. (red) and UV det. (blue)
detection limits for alkaloids are in the 10-7 - 10-8M range (injected) - some 10x better than UV
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Column: Nucleodur CN-RP 5µm 250mmm x 4,6mmFlow : 0,5mL/minEluent: A= 50% CH3CN / 0,1% HCOOH B= 0,1% HCOOHGradient: 10% - 75% A in 90minInj: 20µLUV: 304nmelectrode: PVC body compB 3mmmembrane: TCPB-DOS 40µL
Boldifolium extract (CERTA) 47mg0,5 mL 0,1M HCl0,5 mL CH3 OH2 hours ultrasonefilter: 0,45µm
time: min
POT: mVmAU
Boldine 10-3 M
Boldo leaf extract, crude
red tracing: potentiometric detector
blue tracing: UV detector
40mV
bromhexin
ambroxol
clenbuterol
0 4 min
bromhexin 10-5M inj.
det. lim: 2x10-10 M, 1pg!
HPLC/Potentiometry of Mucolytic drugs
CN 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
One of our first applications on basic drugs: Chromatographia 57 (11-12): 757-765, 2003
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a
b
c
d
e
10mV/S-1
Domperidone (a 38 μM), enilconazole (b 12 μM), ketoconazole (c 12 μM), cinnarizine (d 12 μM), and isoconazole (e 12 μM). Waters Sunfire C18 3.5µm 100x3.5 column. Flow-rate 1.2mL min-1, 10µL injected. From 0.1M HCOOH/20% CH3CN to 0.1M HCOOH/40% CH3CN in 30 min.
10x better detection limits than UV
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10mV/S-1
a
b
cNN
N N
CH3
HH
H
NN
N N
CH3
HH
H
NN
N N
CH3
HH
H
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 NaH2PO4 pH 2.79. 10 µL injected.
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C3
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C5C6 C7
10mV/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 NaH2PO4/5% CH3CN (pH 2.8) to 30% CH3CN in 10 min.
N NH
NH
O
C18H37
NH
NH
O
C18H37
Urea receptor used in the sensor coating
Time, min
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C6 C8 C10 C12
10mV/S-1
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% CH3CN / 10mM Na2HPO4 pH 7, solvent B= CH3CN. From 0% B to 80% B in 30 min. 1mL min-1, 30°C
N NH
NH
O
C18H37
NH
NH
O
C18H37
Extra receptor molecule used in the sensor coating
Time, min
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10mV/S-1
urine sample, male, showing naturally present dehydroepiandrosterone sulfate (DHEAS). Dionex Acclaim Surfactant column (5µm 120A 4.6 x 150mm). Solvent A= 30% CH3CN / 10mM Na2HPO4 pH 7, solvent B= CH3CN. From 0% B to 80% B in 30 min. 1mL min-1, 30°C
Receptor molecule used in coating
NN
N N
C H3
CH3
H
H
Time, min
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10mV/S-1
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uv signal
dT10dT20
dT30
potentiometry signal
rel.units
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
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
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10mV/60
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C4
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mV/ S-1
C3C4
C6
C7
C8C9 C10
NN
N N
C H3
HH
H
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N N
C H3
HH
H
NN
N N
C H3
HH
H
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% CH3CN/20mM NaH2PO4 pH 2.35, B: 65% CH3CN/20mM NaH2PO4 pH 2.35.
(heptafluoro)butyric acid
Ionophore used in membrane
F
F F
F F
FF
O
O
H
Quinine
Clozapine
Cocaine
Noscapine
12 24 time, min
40mV
Sample 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).
Only 1 example in CE is given here (Anal. Chem. 2006)
Conclusions for HPLC
HPLC detection is sensitive (sub micromolar injected concentrations) and accurate.
It is at its best for lipophilic (logP > 2) cationic organics. Anionic organics with sulfate and sulfonic groups, dicarboxylic acids, perfluorinated carboxylic acids.
Also large multiply charged large molecules can be dealt with.
The potential for applications is very high, in industrial-, food- and biotech fields.