potentiometry

analyte is measured based on the electrical potential (voltage) developed by a cell

do not want the measurement to alter the potential, very low current flow

Ag/AgCl

aX (known)

Sat’d KCl

aX (sample)

Ag/AgCl

Sat’d KCl

V

advantages and disadvantages

! fast ! accurate! precise! sensitive to interferents! wide dynamic range (6-8 orders in some

cases)! low detection limits! (usually) single species detection! electrodes have limited lifetimes! inexpensive! simple to use

reference electrode requirements

The reference electrode should provide a stable and reproducible

potential under a wide variety of sample conditions.

The reference electrode filling solution should not interfere with the

sensing electrode. Ions that interfere with the analyses should not be

introduced into the sample by the reference electrode.

(Use a salt bridge to isolate sample from reference electrode.

Double-junction reference electrodes have salt bridge built-in)

The filling solution should flow freely, with no fouling or plugging of

the junction by the sample.

(silver ion precipitates with proteins, sulfide and TRIS buffers

better to use Calomel )

Reference electrodeselectrode that maintains a constant potential against

which the potential of another half cell may be

measured.

Standard Hydrogen Electrode

AgCl Electrode

Saturated calomel Electrode

junction potentials

electrical potential that develops at the

junction between two electrolyte solutions

arises from unequal rates of diffusion of the

cation and anion

K+ and Cl- have almost equal mobilities,

reduces junction potential to ~5 mV but is

ionic strength dependent

ultimately junction potential limits

precision of ISE to 2%

ion selective electrodes- indicator electrode whose potential is selectively dependent on the activity of one particular ion in solution

- key component is a membrane which interacts selectively with one type of ion

- differences in the activity of the selected ion on each side of the membrane lead to a potential difference across the membrane

Ag/AgCl

aX (known)

Sat’d KCl

aX (sample)

Ag/AgCl

Sat’d KCl

V

membrane propertiesLow solubility (~0)

Low conductivity - do not want any significant redox reaction to

occur. This would alter the system being measured. Some current

must flow however in order for measurement to be made.

Selective reaction with an ion in solution. These can be ion

exchange, crystallization or complexation

Solid state (crystalline) membranese.g., LaF3 for F-, AgCl for Cl-, Ag2S for Ag+ or S2-

Liquid membranes - an ion carrier dissolved in an organic matrix. e.g., K+ ISE use the complexant valinomycin in a PVC polymer film

Glass electrodese.g., pH electrodes

interference and selectivityNicolsky equation

Errors in pH measurements

pH measurement is no more accurate that the pH standards which are typically good to 0.01 pH unit. Also, the pH of the standards can vary by as much as 0.01/C. Consult Table 23-6 for the true pH of common standards over a range of temperatures.

Uncertainty in the junction potential limits the measurement of absolute pH to no better than 0.01 pH unit, and better than 0.03 pH units demands considerable care. Particular problems have been noted in low ionic strength samples such as acid rain. However, changes in pH as low as 0.001 pH unit can be reliably detected.

Acid error: At pH < 0.5, glass electrodes yield pH values that are biased high. Reasons for this are not well understood.

Errors in pH measurementsAlkaline error: pH electrode has a small but significant response to Na+ and

other alkali metals. In alkaline solution (pH > 11), the concentration of H+ is very low (see Table above) while the concentration of metal ion may be very high. The electrode then mistakes some Na+ for H+, and as a consequence reports a lower pH value than is actually present.

Dehydration: A dry electrode requires several hours soaking in water before it will respond correctly to pH.

Electrode Fouling: Fouling of the glass membrane is the most common cause of error in any pH measuring system. The sample conditioning system should minimize electrode foaling. However, in some industrial applications, fouling cannot be entirely prevented. As such periodic (gentle) cleaning of the electrode must be performed. Different manufacturers provide a variety of approaches for performing cleaning.

amperometry

monitor the current at a set potential

commonly used for detection in HPLC/CE

FIAneurophysiology

phenols

amino acids

aromatic amines

sugars

carbohydrates

polyamines

polyalcohols

thiols

oxidase enzyme substrates

disulfides

sulfites

peroxides

thioureas

Organic Nitro cpds

common chemistries

In cyclic voltammetry, the current at the electrode is measured while the voltage applied to the electrode is cycled between an upper and lower limit.

The above data for a gold electrode shows that glucose yields the most current at an electrode potential of 150 - 200 mV. This is typical of almost all carbohydrates.

Cyclic Voltammetry of Glucose at a Gold Electrode

-0.1

-0.05

0

0.05

0.1

0.15

-1000 -800 -600 -400 -200 0 200 400 600 800

Electrode Potential (millivolts)

Ele

ctr

od

e C

urr

en

t (m

illiam

ps)

With Glucose

Without Glucose

an undergrad study on amperometric detection of sugarsMark Jensen at Concordia College Minnesota

The data shown at right is for three injections of equal amounts of pure glucose into the chromatography system. The gold electrode was held at 150 mV.

We would expect that each peak would be the same height, but this is clearly not the case.

The problem is that products of the electrochemical reactions of sugars remain on the electrode surface and “poison” it for further reactions.

0

0.5

1

1.5

2

2.5

3

time

ch

arg

e (

mC

)

electrode fouling pulsed amperometric detection

Three-Step PAD Waveform

-0.6

-0.4

-0.2

0

0.2

0.4

0.6

0.8

0 500 1000

time (milliseconds)

Ele

ctr

od

e P

ote

nti

al

(mil

liv

olt

s)

Step 1

Step 3

Step 21. Detection: The current at the electrode is measured and recorded.

2. Surface Oxidation: A layer of gold oxide forms on the electrode and knocks away any foreign material.

3. Oxide Stripping: The oxide layer is removed resulting in a clean gold surface.

A method must be developed to remove these poisons from the electrode surface during detection. For this, we can take advantage of the electrochemical reactions of gold which occur as a function of the applied electrode potential.

A three-step waveform is applied to the electrode approximately once every second. The purpose of each step is as follows:

improved reproducibility of PA

The graph at right shows the results of three successive injections of equivalent amounts of glucose onto the chromatography column.

The three-step PAD waveform was

used for detection of the glucose.

It can be seen that all peaks are identical in both height and shape, as would be expected for injections of equivalent amount of glucose.

LC-PAD is now the most commonly used analytical method for determining carbohydrate levels.

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

time

ch

arg

e (

mC

)

scanning electrochemical microscope

http://www.msstate.edu/dept/chemistry/dow1/secm/secm.html

use an ultramicroelectrode to map electrochemically detectable processes.

usually used with amperometry, but can also be used with potentiomatry and voltammetry

characteristic shape for microelectrode voltammetry

more SECMthe SECM can be used to determine electron transfer

rate constants of a chosen mediator by observation

of the tip current as a function of tip-substrate

separation and by tip, kt, and substrate, ks,electron

transfer rates.

SECM has been used to make high-resolution chemical concentration maps of

corroding metal surfaces, biological materials, and polymeric materials.

In addition, spatially resolved quantitative measurement of ion flux through porous

material such as mouse skin and dental material can be made.

Potentiometric GC mode uses an ISE tip, which has the

advantages of minimizing perturbation of the diffusion

layer, sensitivity to non-electroactive ions, and selectivity

for the imaged ion. Images have been made of pH, Ca2+,

NH4+, etc.

Positive Feedback is illustrated by the following

two animations. The Feedback process

occurs when the mediator is restored to its

original oxidation state at the substrate by an

electrochemical, chemical, or enzymatic

reaction. As the gap width, d, decreases the

regeneration of the mediator tip-substrate gap

becomes more efficient and the current, IT,

increases.

Negative Feedback occurs by the physical

blocking of diffusion of mediator molecules to

the tip electrode by the surface. This produces

a drop in tip current, IT, with a decrease in gap

width, d, as illustrated below.

neuroscience single pore

measure secretion from nerve cells

The cartoon depicts exocytosis of a granule and detection of

secretory products by their oxidation at a Carbon Fiber

Electrode placed nearby, a technique developed by Mark

Wightman and colleagues. The black trace shows quantal

amperometric current spikes due to exocytosis of

catecholamine-containing granules from an isolated adrenal

chromaffin cell stimulated by 100 mM nicotine. Scale = 20 pA

and 2 s. Trace from Wightman, R.M., et al., (1991) Proc. Natl. Acad. Sci. USA 88, 10754-10758.

Image shows an isolated leech neuron (70 um diameter)

with a carbon fiber electrode placed near its tip. The traces

show two amperometric current spikes, quantal responses

due to secretion from a large granule (top trace) and,

possibly, from a small synaptic vesicle (bottom trace).

Scale = 10 pA and 5 ms.

Bruns, D. and Jahn, R. (1995) Nature 377, 62-65.

electrophysiology robot

funetics.com

12 pipettes with force feedback