MLAB 2401: Clinical Chemistry Keri Brophy-Martinez

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MLAB 2401 Clinical ChemistryKeri
Brophy-Martinez

• Analytical Techniques and Instrumentation
• Electromagnetic Radiation Spectrophotometry

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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

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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

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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

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Electromagnetic Spectra
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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

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Electromagnetic RadiationProperties of light
and radiant energy

• Wavelength
• Measured in nanometers (nm) or 10-9 meters.

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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

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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.

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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

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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.

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Beer-Lambert law (Beers Law)
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Beer-Lambert law (Beers Law)

• A 2 logT

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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

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Percent transmittance
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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.

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Photometry/Spectrophotometry

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

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Spectrophotometer / Spectrophotometry

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

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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

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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

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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

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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.

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Spectrophotometer Component

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

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Spectrophotometer Component

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

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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.

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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

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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.

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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

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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.

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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

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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
  .