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