ELECTROANALYTICAL TOOLS FOR (BIO)SENSORS

14.04.04 Wednesday: 1015 - 1200 (Classroom 300, Main Building)

"Amperometric detection on solid state electrodes. General."

Which organic substances can be detected withamperometric sensors and why? How do these sensors work?

- DC Amperometry, Voltammetry

- Potentiometry

- Conductivity

- Tensammetry (double layer capacitance)

- Spectroelectrochemical

Electrochemical sensors:

+++

---

potentiometry

electronics and electrolytics...

(amperometry)

potentio conductivity (Royce Rocklin, Dionex)

amperometric sensors: working principle

1 potentiostat

2 counter electrode

3 working electrode

4 reference electrode

1

23

4

counter electrode

workelectrode

iout

ref. el.

Ework (vs. ref.)

Ew + Er

potentiostat electronics

operational amplifiers as current follower or as voltage follower

amperometry in hydrodynamic conditions

hydrodynamic voltammogram

current

diffusion limited current

e-

O

Rpotential

0 +500 mV vs Ag/AgCl

amperometry in hydrodynamic conditions

Matsuda’s equation for the limiting current il for a wall-jet:

il = cst.c0. Fm3/4.D2/3.γ -5/12.a-1/2.r3/4

current

r

a

il

0 +500 mV

current, i100% O

potential

100% R

0 +500 E, mV

il

E1/2

OR

nEE log059.0

2/1 −=

current, i

il

1 2

0 500 E, mV

(1) Easily oxidizable compound.

(2) More difficult to oxidize compound.

'0

2/1 lnDD

nFRTEE −=

current, i

1 2 3il

0 +500 E, mV

1 fast kinetics

2 slow kinetics

3 background

Most popular electrode materials

Glassy carbon, carbon paste, Pt, Au

B

oxacilline a thiaridazine b narcotine c desipramine d levomepromazine e chloropromazine f carbenicilline g amilaride h triamtereen i codeine j papaverine k amytriptiline l

Aparacetamol a procaine b salicylamide c sulphadiazine d sulphanilamide e diethylstibestrol f oxprenolol g

• Amperometric detection coupled to LC

• Setup in lab

• Home-made large volume wall-jet cell and electrodes

Glassy carbon (left), glassy carbon with coating (right)

0 10 20 min

A classical HPLC application:

catecholamines

1 norepinephrine

2 epinephrine

3 3,4 dihydroxybenzylamine

4 dopamine

5 serotonine

6 5-hydroxyindoleacetic acid

7 homovanillic acid

reversed phase, D.C. amp., glassy carbon, +0.8V(AgCl), phosphate buffer pH 3, gradient 5% to 56% methanol

Is rapid on-line scanning, or the use of multiple electrode systems possible in HPLC, to obtain 3D chromatograms?

low scan-rate

high scan-rate

when scan-rates increase, the "hydrodynamic" character decreases

Amperometry in non-hydrodynamic conditions

When there is no eluent flow, we scan the potential. Most popular: cyclic voltammetry. It tells us how molecules behave in amperometry.

O

OH

COOHH2N

O

O

+ + 2e- + H+

O

O

OH

OH

COOH

NH2

A

C

D

NO2

NHCOCHCl2

OH

OH

chloramphenicol

NO2 NH OH

+ H2O4H+ + 4e- +

NO

+ 2H+ + 2e-

A

B C

2/12/12/10 .)(....45.0 DSRTnFcAFnip =

less common: reductive mode HPLC detection

-500mV(SCE)

benzoquinone

2-OH naphtoquinone

juglonevitamin K

Other characteristics of amperometric detectors:

- Response time (below 1s)

- Coulometric efficiency (around 1% for wall-jet to 100% for porous electrodes)

- Interelectrode variation (36% for GC, n=10, own data)

Is your compound a candidate for DC amperometric detection?

See internet: amperometric (electrochemical), + compound name

See also internet pages with applications, from equipment manufacturers Dionex, Antec, MetrOhm, BAS, Coulochem..

Check it with cyclic voltammetry

Try it with a simple FIA experiment

OH

OH

OH

OH

O O

HO O

OH

HO

phenolics,

hydroxycoumarins, flavones..

HO

O

HO

OEstrogens

Tocopherols

NH2

NH2H2N

H2N SO2NH-RN

H

S

N

R

R-SH

RHO

OH

anilines, benzidines

sulfonamides

indolyl-3

5-hydroxyindoles

phenothiazines

mercaptans

vitamin A, carotenes

O

O

OH

HO

OH

HO

R-SH R-S-S-R

redox status indicators for extra- and intracellular fluids: vitamin c and glutathion

NO2 NH OH

+ H2O4H+ + 4e- +

reductive measurements: aromatic nitro- and nitroso-compounds

HN

OH

+ 2H+ + 2e-

NH2

+ H2O

problems: oxygen

NH2

Advantages of DC amperometric (electrochemical) detection in HPLC:

- Very sensitive. In HPLC: low pg levels

- Linear calibration curves over wide range

- Selective

- Cheap

Disadvantages in HPLC:

- Electrodes are surfaces. Surfaces are variable and require experience in handling

- Reference electrodes and air bubbles can give high impedances.

What about the use of DC amperometry in CE and lab-on-chip?

Tanyanyiwa, J.; Leuthardt, S.; Hauser, P. C. Electrophoresis2002, 23, 3659-66.Some 90references on conductivity plus potentiometry in CE and microchip capillary. Mostly conductivity.

Wang, J. Talanta 2002, 56, 223-3123 applications of amperometry in miniaturized separation systems from 1998 to 2001.

Amperometric sensors and microsensors for use in physiology, biomedical, cell cultures, bioreactors..Examples for NO:

Source: World Precision Instruments, Lab Equipment for the life sciences, catalog 2004. www.wpi-europe.com

Source: World Precision Instruments, Lab Equipment for the life sciences, catalog 2004. www.wpi-europe.com

Sensor surface uncovered Sensor surface covered with permselective membrane

Example: keeping dopamine out with an anion exchanger membrane

small molecules, ions

minerals, bioparticles,..proteins,

oligosacharides, oils in micelles..

colloidal systems like blood contain enzymes which provoke blood clotting (thrombosis) when sensors are inserted.

Is in-vivo monitoring possible with (amperometric) sensors?

No, lipophilic sensor surfaces provoke blood clots within seconds to hours →thromboses.

And matrix too complex! Interferences unless biosensor is used!

Thrombocytes (platelets). Smallest cellular component of blood. 250,000 per cubic mm.They secrete the protein fibrinogen. Platelets, red and white blood cells get trapped infibres.

Solutions eg for potentiometric sensors:

- Use hydrophilic materials for sensors (eg polyurethanes), or use techniques from leech: NO as anticoagulant + anaesthetic

Solutions for amperometric sensors:

- Do sampling with microdialysis, and analysis with µLC and ECD. Or use biosensors (glucose, urea).

membranes

water………….solution………..colloïdal……….suspension

semi-permeable membrane

dialysis membrane

osmosis membrane filter

to HPLC/ECD

microdialyse vanextracellulaire vochten(bloed, planten..)

0,1mm

microdialysis probe