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
V

advantages and disadvantages

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Ag/AgCl Satd KCl

Ag/AgCl Satd KCl

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aX (known)

aX (sample)

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 electrodes
electrode that maintains a constant potential against which the potential of another half cell may be measured.

Standard Hydrogen Electrode

Saturated calomel Electrode

AgCl Electrode

junction potentials

- 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
V

ion selective electrodes

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%

Ag/AgCl Satd KCl

Ag/AgCl Satd KCl

aX (known)

aX (sample)

membrane properties
Low 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) membranes e.g., LaF3 for F-, AgCl for Cl-, Ag2S for Ag+ or S2Liquid membranes - an ion carrier dissolved in an organic matrix. e.g., K+ ISE use the complexant valinomycin in a PVC polymer film Glass electrodes e.g., pH electrodes

interference and selectivity

Nicolsky 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 measurements
Alkaline 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 FIA neurophysiology

an undergrad study on amperometric detection of sugars

Mark Jensen at Concordia College Minnesota
Cyclic Voltammetry of Glucose at a Gold Electrode
0.15

Electrode Current (milliamps)

0.1

W ith Glucose W ithout Glucose

0.05

-0.05

common chemistries
phenols amino acids aromatic amines sugars carbohydrates polyamines polyalcohols thiols oxidase enzyme substrates disulfides sulfites peroxides thioureas Organic Nitro cpds

-0.1 -1000 -800 -600 -400 -200 0 200 400 600 800

Electrode Potential (millivolts)

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.

electrode fouling
3
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.

pulsed amperometric detection

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: Three-Step PAD Waveform
0.8

2.5 2 1.5 1 0.5 0 time

charge (mC)

Electrode Potential (millivolts)

1. 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.

Step 2
0.6 0.4 0.2 0 -0.2 -0.4 -0.6 0 500 1000 time (milliseconds)

Step 1

Step 3

improved reproducibility of PA
The graph at right shows the results of three successive injections of equivalent amounts of glucose onto the chromatography column.
charge (mC)
4.5 4 3.5 3 2.5 2 1.5 1 0.5 0 time

scanning electrochemical microscope

use an ultramicroelectrode to map electrochemically detectable processes. usually used with amperometry, but can also be used with potentiomatry and voltammetry

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.

characteristic shape for microelectrode voltammetry

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

more SECM
the 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.

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 efcient and the current, IT, increases.

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 ux 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.

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 chromafn 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.

electrophysiology robot

12 pipettes with force feedback

Image shows an isolated leech neuron (70 um diameter) with a carbon ber 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.

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