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MLAB 2401: Clinical Chemistry Keri Brophy-Martinez

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MLAB 2401: Clinical Chemistry Keri Brophy-Martinez Analytical Techniques and Instrumentation Electromagnetic Radiation & Spectrophotometry * ... – PowerPoint PPT presentation

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Title: MLAB 2401: Clinical Chemistry Keri Brophy-Martinez


1
MLAB 2401 Clinical ChemistryKeri
Brophy-Martinez
  • Analytical Techniques and Instrumentation
  • Electromagnetic Radiation Spectrophotometry

2
Introduction
  • How do we actually measure the concentrations of
    molecules that are dissolved in the blood?
  • Spectrophotometry

    Mix chemicals together to
    produce colored products , shine a specific
    wavelength of light thru the solution and measure
    how much of the light gets absorbed
  • Nephelometry and Turbidimetry

    Mix chemicals together to produce
    cloudy or particulate matter , shine a light thru
    the suspension and measure how much light gets
    absorbed or refracted
  • pH Meters / Ion Selective Electrodes (ISE)

    Electrically charged ions effect
    potentials of electrochemical circuits
  • Electrophoresis

    Charged molecules move at
    different rates when pulled through an
    electrical field
  • Osmometers

    Dissolved molecules ions
    are measured by freezing point depression and
    vapor pressure

3
Electromagnetic RadiationProperties of light
and radiant energy
  • Electromagnetic radiation is described as photons
    of energy traveling in waves
  • There is a relationship between energy and the
    length of the wave (wavelength)
  • The more energy contained, the more frequent the
    wave and therefore, the shorter the wavelength

4
Electromagnetic RadiationProperties of light
and radiant energy
  • This relationship between energy and light is
    expressed by Planck's formula
  • E hf
  • Where E energy of a photon
  • h a constant
  • f frequency
  • The formula shows that the higher the frequency
    the higher the energy or the lower the
    frequency, the lower the energy
  • We do not use this to perform any calculations.
    You only need to recognize Plancks formula and
    its components

5
Electromagnetic Spectra
6
Electromagnetic Radiation Properties of light
and radiant energy
  • White light
  • Combination of all wavelengths of light
  • Diffract (bend) white light and all the colors
    become visible
  • The color you see depends on the wavelength of
    color(s) that are not being absorbed
  • Light that is not being absorbed is being
    transmitted

7
Electromagnetic RadiationProperties of light
and radiant energy
  • Wavelength
  • Measured in nanometers (nm) or 10-9 meters.

8
Electromagnetic Radiation Properties of light
and radiant energy
  • Interactions of light and matter
  • When an atom, ion, or molecule absorbs a photon,
    the additional energy results in an alteration of
    state (it becomes excited). Depending on the
    individual species, this may mean that a
    valence electron has been put into a higher
    energy level, or that the vibration or rotation
    of covalent bonds of the molecule have been
    changed.
  • Ultimately, as energy is released, an emission
    spectra is formed

9
Electromagnetic Radiation (Properties of light
and radiant energy)
  • In order for a ray of radiation to be absorbed it
    must
  • Have the same frequency of the rotational or
    vibrational frequency in the molecules it
    strikes, and
  • Be able to give up energy to the molecule it
    strikes.

10
Electromagnetic Radiation
  • Many lab chemistry instruments measure either the
    absorption or emission of radiant energy /light.
  • Spectroscopy is based on the mathematical
    relationship between solute concentration light
    absorbance
  • Beers law

11
Electromagnetic Radiation
  • Beer's Law
  • States the relationship between the absorption of
    light by a solution and the concentration of the
    material in the solution.
  • The absorption and/or transmission of light
    through a specimen is used to determine molar
    concentration of a substance.

12
Beer-Lambert law (Beers Law)
13
Beer-Lambert law (Beers Law)
  • A 2 logT

14
Requirements for Beers Law
  • Keep light path constant by using matching sample
    cuvettes standardized for diameter and thickness
  • Solution demonstrates a straight line or linear
    relationship between two quantities in which the
    change in one (absorption) produces a
    proportional change in the other (concentration).
  • Not all solutions demonstrate a straight line
    graph at all concentrations.
  • If these rules are followed, we can calculate /
    determine an unknowns concentration, by
    comparing a characteristic (its absorbance) to
    the same characteristic of the standard (whose
    concentration is known by definition)
  • Concentration unk (Aunk /Astd)
    Concentration std

15
Percent transmittance
16
Photometry/Spectrophotometry
  • In photometry we measure the amount of light
    transmitted through a solution in order to
    determine the concentration of the light
    absorbing molecules present within.

17
Photometry/Spectrophotometry
  • Types -Simple photometers and colorimeters use a
    filter to produce light of one wavelength
    (monochromatic light).

18
Spectrophotometer / Spectrophotometry
  • Spectrophotometers differ from photometers in
    that they use prisms or diffraction gratings to
    form monochromatic light.

19
Spectrophotometer Components
  • Light source/lamps
  • Vary according to need, but must be a constant
    beam, cool and orderly
  • Types
  • Tungsten or tungsten iodide lamps for visible and
    near infrared
  • Incandescent light (400 nm - 700 nm)
  • Deuterium or mercury-arc lamps required for work
    in U.V. range
  • Range 160-375 nm

20
Spectrophotometer Components
  • Monochromators
  • Promote spectral isolation
  • Operator selects specific wavelength
  • Isolate a single wavelength of light
  • Provides increased sensitivity specificity
  • Types
  • Glass filters
  • Prisms
  • Diffraction gratings

21
Spectrophotometer Components
  • Monochromator characteristic
  • Bandpass/bandwidth
  • Measures the success of the monochromator
  • Defines the width of the segment of the spectrum
    that will be isolated by the monochromator

22
Spectrophotometer Components
  • Cuvet
  • Made of high quality glass or quartz
  • Glass for work in the visible light range
  • Quartz or fused silica for work in the UV range
  • Shape
  • Round cuvets are cheaper but light refraction and
    distortion occur
  • Square cuvets have less light refraction but
    usually more costly
  • Optically clean
  • No inconsistencies in composition
  • No marks, scratches, or fingerprints
  • Positioning
  • Orientation and placement into the instrument
    important. Each time must be the same so light
    passes through the cuvet at the same place.

23
Spectrophotometer Component
  • Photodetectors
  • Purpose to convert the transmitted light into
    an equivalent amount of electrical energy
  • Most common is the photomultiplier tube

24
Spectrophotometer Component
  • Readout devices
  • Purpose to convert the electrical signal from
    the detector to a usable form
  • Types
  • Meters/Galvanometers
  • Recorders
  • Digital Readout

25
Spectrophotometer Quality Assurance
  • Wavelength calibration or accuracy is checked by
    using special filters with known peak
    transmission
  • Should be done periodically
  • Must be done if a parameter, such as a change in
    light / lamp has taken place.
  • Must be done if the instrument has been bumped or
    traumatized.
  • Wavelength calibration verifies that the
    wavelength indicated on the dial is what is being
    passed through the monochromator.

26
Spectrophotometer QA
  • Stray light
  • any wavelength of light reaching the detector,
    outside the range of wavelengths being
    transmitted by the monochromator.
  • Spectrophotometers must be periodically checked
    for Stray Light
  • Causes insensitivity and linearity issues
  • Resolve by cleaning optical system

27
Spectrophotometer QA
  • Linearity Check
  • A linearity check is made by reading the
    absorbance of a set of standard solutions
    (obtained commercially) at specified
    wavelength(s), or by using neutral density
    filters
  • Produces a graph similar in appearance to
    standard curve.

28
Spectrophotometer Sources of Error
  • Lamp burnout most frequent source of error
  • Hours of use can be logged by system
  • Watch for lamp to turn dark or smoky in color
  • Monochromator error
  • Poor resolution due to wide bandpass
  • Results in decreased linearity and sensitivity
  • Cuvet errors
  • Dirt, scratches, loose cuvet holder - all cause
    stray light
  • Air bubbles in specimen

29
Spectrophotometer Sources of Error
  • Reagent make-up
  • some test procedures make a product that easily
    foams
  • Volume too low for light path
  • Electrical static (noise)
  • Dark current - from the detector. Leakage of
    electrons when no light passing through.

30
Nephelometer
  • Principle
  • Measures scattered light
  • Light bounces off insoluble complexes and hits
    a photodetector
  • The photodetector is at an angle off from the
    initial direction of the light.
  • This is a measure of Light Scatter
  • Clinical Applications
  • Protein measurements in serum, CSF,
    immunoglobulins, etc.
  • Most of the component parts are similar to those
    of the spectrophotometer.
  • Major differences
  • The position of the detector and
  • reduces stray light
  • Light source/beam LASER light

31
References
  • Bishop, M., Fody, E., Schoeff, l. (2010).
    Clinical Chemistry Techniques, principles,
    Correlations. Baltimore Wolters Kluwer
    Lippincott Williams Wilkins.
  • Sunheimer, R., Graves, L. (2010). Clinical
    Laboratory Chemistry. Upper Saddle River Pearson
    .
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