Intro to spectrophotometry and electronchemistry

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Transcript of Intro to spectrophotometry and electronchemistry

  • Mercury Y. Lin MD FCAP LCDR MC USN

    NHCP

  • Is the measurement of light intensity at a selected wave length

    In clinical lab, radiant energy can be measured based on

    -emission

    -transmission

    -absorption

    -light scatter

    -reflection

  • TSample=IS/Io

    TRef=IR/Io

    So,

    Tcompound of interest = IS/IR.

  • The amount of light absorbed as light passes through is:

    Board Exam Q.!

  • Beers Law [concentration of a substance is directly proportional to the amount of light absorbed].

    b is usually set a 1 cm, and c is in moles/liter. So [Absorbance] [concentration]

  • Absorbance is only directly proportional to concentration when within linearity (range established experimentally).

    Incident radiation on the substance must be monochromatic.

    No chemical reaction occurs. Because of relative concentration errors at

    very high and very low end of absorbance, optical system should be designed to analyze between 0.1 to 0.7 absorbance unit (20% and 80% T).

  • 1. analyte absorbs at a unique wavelenth (uncommon)

    2. analyte reacts with a reagent to produce an adduct that absorbs at unique wavelength.

    3. analyte is involved in reaction that produces chromophore.

  • Example: Total Protein Quantitation. Assays can be designed to look for the same analyte via

    different wavelengths

    Lowry Assay

    Bradford/Coomassie blue assay

    Bicinchoninic acid Assay

    Copper based, classic Biuret reaction

  • Direct Spectrophotometry: Biliruin has maximum absobance @ 455 nm. But oxyhemoglobin absorbs @ 455 nm and 575 nm. (we can mathematically subtract out oxyHb interference).

  • A more common way to measure bilirubin is by Diazo reaction

    Board Exam Q.!

  • There are actually 4 types of bilirubin. -bilirubin: unconjugated -bilirubin: monoconjugated to glucuronic

    acid -bilirubin: diconjugated to glucuronic acid -bilirubin: bound to protein.

    The conjugated forms (,,) are water

    soluble. The unconjugated form () becomes soluble

    when EtOH is present.

  • Now, Diazo reaction is accelerated in the presence of EtOH, So this is how we get:

    #1: Diazo rxn + accelerator => total bilirubin

    #2: Diazo rxn w/o accelerator => direct (conjugated ) bilirubin

    Take #1-#2=indirect (unconjugated) bilirubin

    Board Exam Q.!

  • To ensure optimal function, periodic monitoring is required in:

    - Photometric Accuracy (is the set wavelength accurate?)

    - Absorbance check (compare result with known standards)

    - Linearity

    - Stray light (e.g. light source external to the system).

  • Beers Law loses linearity at the high and low concentration ends of the relationship. For absorbances below 0.1 and above 1.0 considerable errors may be expected (but this will be dependent upon the performance characteristics of the individual spectrophotometer).

  • Atomic Absorption (AA) Spectrophotometry: element of interest is atomized (dissociated from its chemical bond), set to lower energy state, and its ability to absorb radiation is measured. Used to measure aluminum, copper, lithium, lead, Zn, magnesium.

    Flame Emission Spectrophotometry: metal elements are subject to hot flame and its light emission is measured (e.g. lithium=red, sodium=yellow, potassium=violet). Now rarely used.

  • Fluorescence: when a molecule absorbs light at one wave length and reemits at a longer wavelength.

    Flurometry is the measurement of emitted fluorescence light.

    Stokes Shift: the difference between wavelength of excited light vs the wavelength of the emitted fluorescence light.

  • Modifying Beers Law, the relationship of fluorescence emission to substance concentration is

    **This is equation is only valid when the solution absorbs

  • Fluorescence measurements are 100X 1,000X more sensitive than absorbance measurement (due to higher signal and much less noise compared to conventional photometry).

    Flow cytometry is essentially a laser-induced fluorometer + light scatter analyzer.

    Board Exam Q.!

  • Both are methods of measuring light scatter. Mathematic equations show light scatter is

    related to: -particle size -wavelength dependence -distance of observation -polarized incident light source -concentration of particle -molecular weight of particle IMHO, beyond the scope for practicing

    pathologists. What we need to know are:

  • Are optical techniques to measure precipitate formation in Ag-Ab reactions.

    Turbidimetry: is the measurement of intensity of incident light beam as it passes through a solution containing substance of interest.

    Nephelometry: is the measurement of light energy reflected/scattered that is NOT in the direction of the transmitted light. (commonly 90 angle to incident light).

    Generally speaking, nephelometry is more sensitive than turbidimetry with a lower limit of detection (1-10 mg/L for serum protein).

    Board Exam Q.!

  • 1. Antigen Excess: - too much antigens which saturate all available antibody

    binding sites. - during antigen excess, light scattering signal falls

    because of immune complexes lattice are not well formed.

    Board Exam Q.!

  • 2. Matrix Effect: to some extend, all molecules scatter light. Matrix is all components of a material system, except the substance of interest(analyte).

  • Involves the measurement of current or voltage generated by the activity of specific ions.

    4 analytical subtypes of electrochemistry utilized in clinical labs are:

    -Potentiometry

    -Voltammetry/Amperometry

    -Conductometry

    -Coulometry

  • Measures potential (voltage) between 2 electrodes (half-cell)

    is widely used in measurement of electrolytes and blood gas.

    An electrode with a constant (known) voltage is termed reference electrode. This electrode is insensitive to the substance in the solution that is being studies. Examples of reference electrodes used in clinical labs include: calomel (HgCl)electrodes and Ag/AgCl electrodes.

    The meauring electrode is termed indicator electrode.

  • The concentration of ion in a solution is then calculated from the potential between 2 electrodes, using Nernst Equation.

    E = E0 (0.0592/n) log Cred/Cox

    Cell potential measured

    Standard reduction potential

    Number of electrons involved in reaction

    Concentration of reduced species

    Concentration of oxidized species

  • In Ion selective electrodes(ISE), either glass or polymeric membranes are utilized to enables optimal permselectivity of ion of interest.

    Glass electrodes are used in H+ and Na+ measurements.

    Polymer membrane electrodes are available for K+,Ca2+,Li+,Mg2+,bicarb measurements.

  • Valinomycin: selective for K+. Board Exam Q.!

  • Direct ISE:

    - measures analyte in undiluted whole blood or plasma sample.

    - typically used in blood gas analyzers and POCT electrolyte analyzers (But also used in OCD Vitros systems).

    - no centrifugation needed if using whole blood.

  • Indirect ISE:

    - measures plasma or serum that has been preanalytically diluted with a diluent.

    - requires that the plasma and erythrocytes are separated by centrifugation.

    - typically used in high volume centralized laboratory chemistry analyzers.

    - generally comparable to flame photometry (the reference method).

  • The principle of potentiometry is identical in both direct ISE and indirect ISE.

    However, disagreement can arise in clinical situations. Example: - Normally, serum is 93% water phase and 7% nonaqueous

    phase. Na is located in serum water phase only. - If the nonaqueous phase is expanded (e.g.

    hyperlipidemia or hyperproteinemia), the water phase % is decreased.

    - so less Na is present in a given volume of serum (even though [Na] in the water phase is unchanged] results in pseudohyponatremia. The phenomenon is seen in Indirect ISE systems.

  • Measures the quantity of electricity (in coulombs) required to convert an analyte to a different oxidation state.

    1 coulomb = quantity of electricity that is transported in 1 sec by a 1 amp current.

    Coulometry is used to measure [Cl].

  • To measure chloride, a constant current is applied to 2 Ag electrodes => which liberates Ag+ at a constant rate.

    Cl- in solution combines with Ag+ to form insoluble AgCl. [Basically: a silver ion for every chloride ion].

    Once there is excess Ag+, the reaction stops.[see Amperometry on how the systems knows when to stop]

    The # of liberated Ag+ = # of Cl- in the solution. Which is calculated using Faradays Law.

  • Amperometry measures the current produced by an oxidation-reduction RXN.

    This is *part 2* of [Chloride] measurement system. As Cl- are consumed and excess Ag+ are in the system, there will be a increase in current. And the endpoint is reached.

    Amperometry is also used to measure pO2 in blood gas. In this setup, polypropylene membrane on the pO2 electrode allows dissolved oxygen to pass through where it reacts with platinum electrode and is reduced. The change in current is proportional to partial pressure of oxygen in the biologic specimen.

  • In voltammetry, potential is applied to an electrochemical cell and