electroanalytical tools for (bio)sensors 14.04.04 ... · advantages of dc amperometric...
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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