Achem Exam 3

electrochemical techniques + mass spec

Coulometry

Counts the electrons used in a reaction under constant current or voltage

Amperometry

Current between a pair of electrodes is measured. One of the reactants is the analyte and the current is proportional to the concentration of the analyte.

• ex) glucose monitor: current is proportional to H₂O₂ which is proportional to glucose

Controlled Potential Electrolysis

Voltage difference between working and reference electrodes in a 3 electrode system is measured and regulated by a potentiostat

Potentiometry

measure voltage difference between 2 electrodes in the absence of current flow.

Cyclic Voltammetry

Current is measured as different voltages are scanned. Can obtain:

• Redox potential

• diffusion constant

• concentration of electroactive species

• number of electrons transferred

• reaction mechanism

• impurities/side reaction

• stability and electrochemical window

• adsorption and surface phenomena

• reaction dynamics like reversibility or kinetic barriers.

Function of each portion of a LFA

Sample Pad: location where sample is applied

Conjugate Pad: holds labeled antibodies or antigen

Nitrocellulose membrane: contains the test and control lines; test line contains immobilized antibodies that capture the labeled analyte complex while control captures excess labeled conjugates to confirm the test worked.

Absorbent Pad: draws the liquid through the strip through capillary action

Backing Card: Provides structural support.

Nominal Mass vs monoisotopic mass

Nominal: integer mass of the species with the most abundant isotope

Monoisotopic: Exact mass of the species with the most abundant isotope.

What is peak area proportional to?

Ionization efficiency and abundance

Hard Ionization Sources

Electron Impact Ionization: sample can be easily vaporized without decomposing, volatile, thermally stable (hydrocarbons, alcohols, esters, etc)

Inductively Coupled Plasma: Sample needs to be analyzed for elemental composition, not structure, best for liquid samples/ metals

Soft Ionization Sources

Chemical Ionization: Reactive molecular ions collide with analyte and ionize them → useful got molecular weight determination of volatile organics

MALDI: Sample mixed with matrix, dried, then pulsed with laser so the matrix will carry sample and charge into gas phase → large biomolecules (proteins, peptides, DNA), not volatile/thermally stable or stable in solution (can’t vaporize)

Electrospray Ionization: zap with electricity to create an aerosol → good for proteins/large thermally liable biomolecules, used with LC-MS, produces multiply charged ions

DESI: Charged droplets are sprayed directly onto a surface and extract the analyte → direct analysis of surfaces under ambient conditions

Reasons why analytes give multiple peaks

• Multiple charged states: Peaks are far apart and divisible by integers

• Isotopic variants: Peaks differ by mass of a neutron

• Multimerization: Peaks differ by molecular weight times an integer

• Adduct formation: Mass of molecule plus adduct (H⁺, Na⁺, Cl^-,H₂O, NH₄⁺)

• Fragmentation: Molecular ion may not be the base peak.

How LFA work

1. Sample is added to the sample pad, which conditions the sample.

2. Sample moves into the conjugate pad and rehydrates the AuNP-antibody conjugates, which can then interact with the target analyte.

3. Analyte (if present) binds to the AuNP-antibody and forms the AuNP-antibody-antigen complex

4. mixture moves along the nitrocellulose membrane

5. At the test line, immobilized antibodies recognize a different epitope on the analyte, capturing the AuNP complex (sandwich assay)

6. At the control line, anti-species antibodies that bind to the AuNP-Antibody complex at the Fc region are present to confirm the test worked

• Stronger line corresponds to more analyte (sandwich)

• Weaker line corresponds to more analyte (competitive_

What is Overpotential

The voltage needed to overcome the activation barrier for transferring an electron.

Ohmic Potential

The voltage needed to overcome the resistance of a solution

Concentration potential

Accounts for differences between the concentration in bulk solution and at the electrode surface.

Ion Selective Electrode

Selectively responds to one ion, doesn’t involve redox processes, contains an ion-selective membrane that selectively binds and transports one ion.

• Diffusion of the analyte ions out of the membrane creates a slight charge imbalance between the membrane and solution

• Changes in the analyte concentration in the solution changes the potential difference

• Use calibration curve to determine analyte concentration