Analytical chemistry electrochemistry

electrochemistry measures

Ph, pCO2, pO2, and several ions

Electrochemistry applies to the

movement of electrons from one compound to another

electrochemical potential is the

driving force behind the electron flow, determining the direction of the electron flow

What is a current

The amount of electrical flow per time (electrons/sec)

What is potentiometry 

measures the difference in electrical potential (volts) between two electrodes 

What is amperometry

Measures the flow of electrical current (ampere)

What are the two different electrodes in potentiometry

Reference electrode

Indicator electrode (test electrode)

What is the reference electrode in potentiometry

baseline potential established

Three types of Reference electrodes in potentiometry

Hydrogen (standard, saturated calomel, Silver/silver chloride)

What does the indicator electrode (test electrode) measure 

Potential changes, which are dependent on the activity (concentration) of analyte

Common indicator electrodes are

ISEs (ion selective electrodes)

How do ISEs work in potentiometry

Uses direct potentiometric measurements, measuring the activity of one ion more than the others 

Are ISEs selective or non-selective

Selective (not specific) for an ion, due to selective ionophores in the membrane of the electrode 

Ion concentration (C) can be calculated from ion activity (a) through the equation

a=yc

What does the internal solution in an Ion-Selective Electrode (ISE) contain?

It contains the ion of interest at a constant activity.

What important component is built into an Ion-Selective Electrode?

An internal reference electrode.

How does an ISE determine ion concentration (Step 1)?

It detects the altered electrical potential (Ei) across a selective membrane caused by the difference in ion activity between the internal solution and the external tested solution.

How does an ISE determine ion concentration (Step 2)?

By comparing the measured potential (Ei) to the fixed standard potential (Er) of a reference electrode.

Electrical potential difference is measured

ΔE = Ei – Er

How does an ISE determine ion concentration (Step 3)?

A calibration curve is created using standard solutions, plotting ΔE vs. Δlog(concentration)

The sensing part of ISE is an 

Ion-selective membrane 

Types of ion selective membranes

Glass membranes - responsive to univalent cations

Polymer membranes

What are the three polymer membranes and what do they measure

Crystalline (solid state): selects primarily anions
Liquid: measures cations (mono & polyvalent) &
some anions

Gas sensor: measures gases

What does a pH glass membrane electrode measure?

Measures hydrogen ion concentration [H+][H⁺][H+] — reported as pH.

What type of electrode is the pH electrode considered?

It is the best-known ion-selective electrode (ISE).

What internal reference does a pH electrode contain?

A silver wire coated with AgCl₂ immersed in internal HCl solution

What makes the pH electrode selective for hydrogen ions?

A special glass membrane tip that responds specifically to H⁺ activity

What is the typical external reference electrode used with pH electrodes?

Either a calomel electrode or an Ag/AgCl electrode

How is electrical potential generated in a pH glass electrode?

Movement of H⁺ ions near the glass membrane creates a potential at the indicator electrode.

How is pH determined by the instrument?

A voltmeter measures the potential difference between indicator and reference electrodes, then converts that potential into pH.

How is the Na⁺ glass membrane electrode different from the H⁺ electrode?

Different glass composition, but has cross-reactivity with H⁺.

What is the selectivity order for a pH (H⁺) glass membrane?

H⁺ >>> Na⁺ > K⁺

What is the selectivity order for a Na⁺ glass membrane?

H⁺ > Na⁺ > K⁺

What forms the ISE membrane in a chloride electrode?

A pellet of AgCl + Ag₂S.

How do Cl⁻ ions generate signal in a solid-state electrode?

Cl⁻ diffuses into crystal lattice vacancies → changes membrane conductivity.

What ions can interfere with a chloride electrode?

F⁻, Br⁻, I⁻.

What provides selectivity in liquid polymer membrane electrodes?

Ionophores in a water-insoluble solvent or PVC.

What common ions do polymer membrane electrodes measure?

K⁺, Na⁺, Ca²⁺, Li⁺, Mg²⁺.

What ionophore is used in a Na⁺ polymer membrane electrode?

Monensin

What is monensin similar to in structure and function?

An antibiotic that selectively binds Na⁺.

What ionophore is used in a K⁺ membrane electrode?

Valinomycin

Why is valinomycin highly selective for K⁺?

It has a pore size matching the unhydrated radius of K⁺ and binds K⁺ reversibly and selectively.

What provides selectivity in a Ca²⁺ polymer membrane electrode?

A cation-exchanger resin impregnated with phosphate ions

How is Ca²⁺ concentration detected in the membrane?

Ca²⁺ binds selectively, generating a membrane potential proportional to Ca²⁺ levels.

What type of electrode is a pCO₂ electrode?

A gas-sensing modified pH electrode.

What membrane property allows pCO₂ measurement?

A CO₂-permeable polymer membrane that allows only CO₂ to diffuse

How is CO₂ converted for measurement?

Sample is acidified → all HCO₃⁻ → CO₂

What is actually measured inside a pCO₂ electrode?

H⁺ from CO₂ hydration, measured by an internal pH electrode (indirect measurement).

What other gases are measured using similar electrodes?

NH₃ and SO₂

Advantages to ISEs

Color or turbidity does not affect results

Sources of Error with ISEs

“dirty electrodes”

Ion selectivity

electrolyte exclusion effect

How does a dirty electrode cause errors in ISEs

Protein coating over membrane causing a decrease in sensitivity and selectivity

How does ion selectivity affect ISEs

Lack of ion selectivity —> Interfering ions 

Ex: Br, F, I

How does the Electrolyte exclusion effect affect ISEs 

Direct—> No dilution: No electrolyte exclusion effect

Indirect—> Sample diluted : Prone to electrolyte exclusion effect 

In the electroylte eclusion effect what yields false lows

Lipemic or high protein samples

Hyperlipidemia or hyperproteinemia —> False low electrolyte values 

What are the two amperometric electrodes

pO2 gas sensing electrodes

Glucose gas sensing electrodes 

What is another name for the pO₂ electrode?

Clark electrode or polarographic electrode.

What is the cathode in a pO₂ electrode?

A platinum wire.

What is the anode in a pO₂ electrode?

Ag/AgCl.

What electrical potential is applied in a pO₂ electrode?

–0.65 V

Will current flow in a pO₂ electrode without oxygen present?

No — O₂ must be present to allow conduction.

How does oxygen reach the cathode in a pO₂ electrode?

O₂ diffuses through a selectively permeable membrane to the cathode.

What happens to O₂ at the cathode?

It is electrolytically reduced, using electrons from the silver anode.

What does the electrical current represent?

Current is proportional to the pO₂ in the sample.

What type of electrode is a glucose electrode technically?

A pO₂ electrode (indirect glucose measurement).

What diffuses into the enzyme layer of a glucose electrode?

Glucose, through a glucose-selective membrane.

What enzyme is immobilized in the glucose electrode?

Glucose oxidase.

What reaction occurs in the glucose electrode?

Glucose + O₂ → Gluconolactone + H₂O₂

How does the glucose electrode measure glucose concentration (Method 1)?

By measuring decreased pO₂ during the glucose oxidase reaction (amperometric).

How does the glucose electrode measure glucose concentration (Method 2)?

By measuring increased H₂O₂ production during the reaction (amperometric).

Blood Gas analyzers uses

Direct measurements

4 electrodes 

1. pH electrode-glass electrode-ISE-potentiometric
2. PCO2 (combination)-membrane electrode-ISE-
potentiometric
3. PO2 (combination)-membrane electrode-
amperometric
4. Reference electrode

What condition is diagnosed using chloride measurement by coulometry?

Cystic Fibrosis (CF) — via sweat chloride.

What instrument is used in coulometric chloride titration?

Cotlove chloridometer.

How is Ag⁺ generated in coulometric titration?

By oxidation of a silver (Ag⁰) wire at the generator anode under constant current.

What reaction occurs between chloride and silver ions?

Ag⁺ + Cl⁻ → AgCl(s) (titration)

What signals the endpoint of the titration?

After all Cl⁻ reacts, excess Ag⁺ is reduced, causing current to rise → timer stops

What is directly proportional to chloride concentration in the sample?

Charge used or titration time

What ions interfere positively with coulometric chloride titration?

Other anions/electronegative radicals such as:
Bromide (Br⁻), Cyanide (CN⁻), and Cysteine.

What is electrophoresis

migration of charged particles in an electrical
field toward the electrode w/ the opposite charge

What is zone electrophoresis

components are separated into distinct & stable zones or bands in a supporting medium

Electrophoresis is used in clinical labs to

separate and measure molecules based on size, charge, & conformational differences

Electorphoretic samples include 

Serum, urine, CSF, blood and other fluids 

Native electrophoresis

Aka non-denaturing

Seperates molecules based on charge, density, size, and shape

Mobility of a molecule is directly proportional to its charge and inversely proportional to its size 

Isoelectric focusing

Seperates molecules according to their isolectric points using immobolized pH gradients 

In isoelectric focusing as proteins electrophorese across an electric field, they

will stop at a position in the gel where the pH is equal to
their pI

Isoelectric focusing is used for special studies like

CSF ologoclonal banding and to determine a proteins pl

In SDS PAGE the sample is pretreated with 

SDS

mobility of a molecule is inversely proportional to size 

In two dimentional electrophoresis the molecules are seperated in a complex micture with two dimensions 

First dimension : IEF

Second dimension : PAGE 

Electrophoretic system 5 components

1. Power supply- provides a constant current
2. Separation media-

agarose gel- which is widely used in native electrophoresis- seperating mainly by charge- good for large molecules 

Polyacrylamide gel (PAGE)- seperates by size- good for small molecules 
3. Sample- Serum, blood, (urine and CSF- need to be concentrated)
4. Buffer-

-Maintains a constant pH most often 8.6

-provides ions to maintain conductivity 
5. Detection, identification & quantitation system- 

dye (visualization),
standards (identification) & densitometer (quantitation)

In an electrophoretic system Direction and migration rate are dependent on

Direction : Charge, depends on Ph and pl

Migration rate: Charge density, depends on difference between Ph and Pl

Steps in classical electrophoresis

1) saturated with an alkaline buffer

2) Constant voltage or current is passed through the medium

3) proteins are stained

4) proteins are identified by comparing their location against standards 

5) Proteins are scanned w/ a densitometer to quantitate their conc.

Is electrophoresis qualitative or quantitative

Both

What factors affect migration (seperation) and are they inversely proportional or proportional ?

1) strength of electric field- directly proportional to voltage

2) properties of a supporting medium- charge and size

3) characteristis of the molecule - directionly proportional to net charge/charge density - inversely proportional to size

4) temperature- proportional to temperature 

5)ionic strength and pH of the buffer - inversely proportional to ionic strength buffer- migration rate increases in a more alkaline buffer

6) endosmosis- slows the migration of large and less neg. charges molecules 

common applications for electrophoresis in clinical chemisty

polyclonal gammopathy

monoclonal gammopathy

What does the presence of an M band or M spike indicate?

Overproduction of a single monoclonal immunoglobulin and its light chain (paraprotein) — suggestive of Multiple Myeloma

Where is the M band located on a serum protein electrophoresis (SPEP) gel?

In the γ (gamma) region.

Where is the M spike located on an electrophoretogram?

In the γ (gamma) region of the SPEP tracing

What test quantifies the size of the M band?

Densitometric scanning of the SPEP.

What follow-up testing is used to identify the type of paraprotein?

Serum and urine immunofixation electrophoresis (IFE).

What is a paraprotein?

A monoclonal immunoglobulin and/or free light chain produced in MM.