Electrochemistry Part 2 PDF

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Summary

- Electrochemistry is used frequently in the clinical lab. - Blood Gas Analyzers, all critical care laboratories have a blood gas analyzer. Measures pH, pCo2 and pO2 directly. - Electrolyte Analyzers, routine tests in a lab performed on general chemistry analyzers. - Metabolite Analyses, glucose, la...

Description

MLSC-3111, Clinical Biochemistry II -

Electrochemistry is used frequently in the clinical lab. Blood Gas Analyzers, all critical care laboratories have a blood gas analyzer. Measures pH, pCo2 and pO2 directly. Electrolyte Analyzers, routine tests in a lab performed on general chemistry analyzers. Metabolite Analyses, glucose, lactate, urea and creatinine.

ISE Polymer Membranes -

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Liquid and Polymer Membrane Electrodes, electrodes that have a liquid consisting of a water-soluble viscous solvent in which is a dissolved ionophore. Also has a thin, porous membrane such as cellulose acetate or PVC (polymers) that the liquid can soak into. Ionophores, hydrophobic organic ion-exchangers that react selectively with an ion (analyte). These are soaked into the PVC membrane to act as chelating agents. They extract target molecules out of solution to incorporate them into the ionophore. All ionophores have cavities of various sizes in their structure to specifically chelate certain ions by size. Several types of ionophores exist that bind cations to membranes. Extraction of the ion causes a positive potential at the membrane interface. The difference can be measured since it is related to the ion activity and therefor the ion concentration in solution. Increased potential means increased activity. The inner solution in the electrode remains constant due to a standard solution. The only potential change measured in the circuit is from the potential at the membrane surface that is in contact with the sample. Potassium ISE, uses an antibiotic as the ionophore (Valinomycin) because it exhibits excellent selectivity for potassium over sodium. The sodium molecule is too small to fit into the Valinomycin ring unlike the perfectly sized potassium. This is relevant because there is so much more sodium in the blood than potassium.

ISE Solid State Membranes -

Consist of single crystals or pressed pellets of salts of the ion of interest mounted at the end of the electrode. The membrane potential is generated by a selective surface reaction between the ion of interest and the crystal on the electrode made of the same ion. Ions in solution are incorporated into the crystal lattice structure on the end of the electrode. Used for fluoride and chloride testing.

VITROS ISE Technology -

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The VITROS does potentiometric testing using PM slides and an incubator. Na+, K+ and Cl- use this method, as it cannot measure TCO2. Each slide has 2 identical electrodes embedded in it. Each slide has a paper bridge with 2 drop holes, 2 identical ISE electrodes and 2 electrical contact areas. Equal amounts of sample and Electrolyte Reference Fluid (ERF) are simultaneously dropped onto the drop holes. The drops flow toward each other along the paper bridge

MLSC-3111, Clinical Biochemistry II

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to form a junction. The sample and ERF have different ionic concentrations, resulting in the creation of 2 half-cell potentials. The potentials are then converted into concentrations. Electrometer, uses 2 pairs of contact points which penetrate the contact areas to allow the measurement of the potential difference between the ERF and the sample. Does not require washing between assays and has no crossover because of the use of a single slide containing the whole cell.

ISE Gas Sensing Membranes -

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Gas Sensing electrodes, consists of an ISE in contact with a thin layer of aqueous electrolyte behind an outer membrane that is permeable to gas of interest. The pCO2 assay uses a modification of the pH electrode. PCO2 Electrode, uses a gas sensing membrane and electrolyte layer that are added to the end of a pH electrode. The pH electrode is covered with a Teflon membrane permeable to CO2. Between the inner and outer membrane of the electrode there is an electrolyte layer made of a weak sodium bicarbonate solution. The CO2 forms carbonic acid and uses the carbonic acid buffer system seen in the body to cause a change in pH. The change in pH can be detected at the inner membrane (which is a pH electrode) as potential which is converted into the pCO2.

Conductance (Voltammetry) -

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Technique in which a potential is applied to an electrochemical cell and the current is measured. Variable potential applied to the cell is controlled by a potentiostat. Voltammetry measures concentration, not activity and the analyte of interest is partially consumed in the reaction. Conductance, the ease of which a current can flow through a medium. Also a form of voltammetry used in clinical labs. It is an electrochemical reaction driven by constant potential resulting in a measurable current which is measured using an AMP meter. The current is directly proportional to the concentration of the analyte of interest. Uses a reference and an indicator electrode plus a membrane selective to the analyte. pO2 Electrode, also known as the Clark Electrode. It has a thin, polymeric, selectively gas-permeable membrane for O2. The inner solution is made of a buffered solution containing NaCl or KCl. Exactly 0.65V is applied between the platinum cathode and the Ag/AgCl anode. The change in current is measured since it is proportional to the analyte concentration. Once current is applied, electrons are generated at the AG/AgCl anode as the anode gives up electrons. Electrons will move to the cathode to produce a current. O2 from the sample diffuses across the outer membrane into the electrolyte solution where it picks up an electron at the cathode. Removal of electrons generates a larger potential and therefore a change in the current. The more oxygen present, the more electrons that are taken up. This makes the oxygen

MLSC-3111, Clinical Biochemistry II

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concentration proportional to the current. However, the oxygen does not go back into solution and is consumed by the electrode. Sample is put into the waste after testing. Polarography, the measurement of the gain/loss of electrons in an electrochemical reaction. The pO2 electrode is a polarography.

Sources of Error and Interferences -

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There are two types of Ise measurement schemes in use: o Direct ISE, measures iconic activity directly in an undiluted sample. o Indirect ISE, pre-dilutes the sample prior to contacting the electrode membrane. Indirect is better because diluting the sample reduces the amount of protein and other blood components that may damage the electrodes. Dilutional effects lead to changes in the sample matrix and indirect ISE activity is influenced by the dilution of protein content. Therefore, the direct ISE is calibrated and adjusted to match the results of the indirect ISE. Indirect ISE’s are most common in the lab, but are also more likely to cause fatal error.

Displacement of Electrolytes -

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Normal plasma is 93% water and 7% solids. Electrolytes are dissolved only into the water phase. This is what the body responds to and is clinical relevant. We report Na+ in mmol/L of plasma which is only 93% water. Lipemic samples will decrease the portion of water in the sample, causing lipids and proteins to displace Na+ when aliquoting the sample. Causes falsely low sodium results (pseudohyponatremia). Electrolyte Exclusion Effect, the exclusion of electrolytes from the fraction of the total blood plasma volume that is occupied by solids. Even in normal samples, there are still proteins to contribute to this effect, but through calibration this effect is eliminated. Lipemic samples would still be falsely low due to lipids excluding the accurate volume of Na+ from being added to the test. Most chemistry analyzers use indirect ISEs, but the BGA and POC analyzers use direct ISEs. Indirect ISE measurements correlate with Flame Emission Spectrophotometry results since they also have this effect. Lipids and proteins can cause this in clinical conditions that cause high levels in either of them.

Other Interferences -

Glass and Ion Exchange Electrodes, interferences are “mistaken” by the electrode for the ion of interest, causing falsely elevated results (positive error results). Solid State Electrodes, the interferent reacts chemically with a constituent of the crystal membrane (poisonous membrane). Can be a positive error, negative error or electrode failure.

MLSC-3111, Clinical Biochemistry II -

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Method interference may be caused by any species in the sample solution that precipitates, complexes, oxidizes or reduces the ion being measured. pH Electrode, may lose sensitivity at pH above 10 due to interference of monovalent cations like Na+. Not a concern in the clinical lab because no one can have a blood pH that high. Na+ Electrode (Glass or Polymer Membrane), polymer electrodes exhibit a false positive in the presence of Lithium (chemically like Na+). Check the patient medications if they have unexplained high Na+ results. Chloride Electrode, contaminated by certain proteins in the sample, but mainly affected by other Halides. Br- is the most common source of Cl- interference in all methods. Temperature, the Nernst equation is dependent on temperature and it must stay constant. Bubbles/Wrinkles in the Membrane, results in imprecision and occurs most often after re-membraning. Protein/Salt Buildup, erratic performance will occur causing response errors. Solved by cleaning the membranes.

Calibration Procedures -

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pH Electrode, uses two specific buffers with approximate values of 6.865 buffer (zero or low point calibrator) and 7.398 buffer (slope or high point calibrator). Each buffer is injected into the same sample chamber, one at a time. pCO2 and pO2, uses two specific concentrations of CO2 and O2 divided into two tanks. o Tank One, low CO2 (balance), high O2 and balance nitrogen. o Tank Two, high CO2 (slope), O2 (0%) and balance nitrogen Two point calibrations are required every 4 hours and 1 point calibrations are required between samples, minimum every 2 hours.

Calibration Parameters -

Slope, the slope of the calibration line has changed. Slope errors may be due to electrode contamination. Intercept, point at which the calibration line crosses the y-axis has an absolute change causing positive or negative bias. Drift, a significant change in measured potential/current between calibrations. Drift error indicate electrode instability, requiring repair or replacement.

MLSC-3111, Clinical Biochemistry II Electrode Maintenance and Handling -

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All electrodes are very fragile and expensive. The electrode function can usually be restored by cleaning their membrane surfaces. The tip must also be maintained in a moist environment. Protein buildup should be cleaned with pepsin, bleach or 0.1M Cl. Inorganic deposits can be removed with EDTA or acidic cleaning solutions. Electrodes can be disposable or reusable (re-membraning). Never touch membranes surfaces with hands. Follow manufacturers instructions on replacing a membrane. The final product must be clean and free of wrinkles/tears. The life expectancy of membranes and disposable electrodes varies per analyte/manufacturer. Periodic cleaning of the pO2 electrode tip with pumice is required because its membrane attracts protein.

Biosensors -

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An analytical device which converts a biological response into an electrical signal. Adds a biochemical reaction layer to an existing electrode. Electrodes measure ion activity or concentration of inorganic analytes. Biosensors use electrodes to measure organic analytes. Most commonly use enzymatic reagents with aerometric transducers. Enzyme catalyzes a reaction involving the analyte of interest and either a product is formed, or a reactant is consumed. The transducer detects the change. Biosensors that use enzymatic recognition of elements are called enzyme electrodes. Glucose Biosensor, consists of a pO2 electrode coupled with the Glucose Oxidase reaction. Referred to as an amperometric electrode, an enzyme electrode or the polarographic method for glucose analysis. The GO solution is physically entrapped between 2 dialysis membranes at the end of a pO2 electrode. Glucose and oxygen can pass through membranes, not proteins. Upon entering the membrane, glucose is oxidized by glucose oxidase. The concentration of O2 will decrease as glucose is oxidized. The decrease in the oxygen concentration is reflected as a decrease in current which can be correlated to a glucose concentration. The membrane is impermeable to all traditional species that would cause interference to the colorimetric GO reaction....