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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5000Pot extract Pot Boldin Uv

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

C4

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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time, min

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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t, min

10 -1

C4

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t, min

mV/ S-1

C3C4

C6

C7

C8C9 C10

NN

N N

C H3

HH

H

NN

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.