Instrumentations - Clinical Chemistry


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(c) Dr. Francis Ian Salaver (soon)

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Instrumentations - Clinical Chemistry

  1. 1. Analytic Techniques and Instrumentation Part 1
  2. 2.  Analytic techniques and instrumentations provide foundation for all the measurements made in the modern clinical chemistry.
  3. 3.  The principles that govern the analytic techniques and instrumentations in the lab fall in to four basic areas:  1. Spectrophotometry  2. Luminescence  3. Chromatography  4. Electroanalytic Methods
  4. 4.  described as photons of energy traveling in waves. These photons or packets of energy have unique frequencies.
  5. 5.  the distance of two peaks or trough when light travels in a wavelike manner The distance between two identical parts in consecutive waves
  6. 6.  Amplitude- the distance between two adjacent peak and trough Frequency- the numbers of waves that pass an observation point in a unit of time
  7. 7.  Ultraviolet- light whose wavelength falls below the visible region (190-340 nm) Visible- light whose wavelength falls within 340-700 nm Infrared- light whose wavelength is above 700 nm
  8. 8.  Always remember that the wavelength is inversely proportional to the frequency of the light wave. Energy is also inversely proportional to the wavelength of light
  9. 9.  The closer the two peaks are, the shorter the wavelength. The shorter the wavelength, the larger number of photons will be contained in a given distance. More photons represents more energy, hence, shorter wavelengths represent higher energy.
  10. 10.  X-ray and Gamma radiations- contain wavelength shorter than 190 nm. These photons have such energy that they can already penetrate the flesh
  11. 11. Wavelength (nm) Color Complementary Color 350-400 Violet Yellow 400-450 Indigo Yellow 450-500 Blue Orange 500-550 Green Red 550-600 Yellow Indigo 600-650 Orange Blue 650-700 Red Green
  12. 12.  Each colored solution absorbs a unique pattern of wavelengths. In general, the perceived color of a solution will be dominated by the wavelength transmitted or emitted by the solution. The transmitted light and the absorbed light are complementary to each other. Thus, a visible color of a solution will be the complement of the wavelength being absorbed.
  13. 13.  A red solution containing hemoglobin will absorb green light at 540nm A yellow bilirubin will absorb __________ at ______nm
  14. 14.  is the measurement of the light transmitted by a solution to determine the concentration of the light-absorbing substance in the solution
  15. 15.  Exciter lamp provides electromagnetic radiation as visible, infrared, or UV light.  Tungsten/Tungsten-iodide lamp or Halogen Quartz lamp  Mercury Vapor Lamp  Vapor lamps and Deuterium Discharge Lamp  Infrared Energy Source ▪ Merst Glower- uses an electrically heated rod of rare earth element oxides ▪ Globar- uses silicon carbide heated to 1200 degree Celsius
  16. 16.  reduces stray light and prevents scattered light from entering the monochromator.  if the stray light, if allowed to pass through the analytical cell, would cause a deviation from the readings
  17. 17.  device that produces light of specific wavelengths from a light source  Monochromatic light- light radiation of a single wavelength
  18. 18.  Types of monochromator  Prism- wedge-shaped piece of glass pieces of glass, quartz, or sodium chloride or some other material that allows transmission of light  Diffraction Gratings- aluminized surfaces that has been cut into tiny grooves that can act as a prism and a slit
  19. 19.  Types of monochromator  Interference Gratings- made by placing silver films on both sides of a dielectric field such as magnesium fluoride. When light passes through the silvered surface, it passes through the dielectric field and is reflected from the second silvered surface back to the first surface  enhances desired wavelength by constructive interference and eliminates others by destructive interferences (thickness of magnesium fluoride)
  20. 20.  Analytical cell Used to hold the solution in the instrument whose concentration is to be measured. Types of cuvettes:  Borosilicate glass cuvette- for alkaline solutions that do not etch glass  Quartz or plastic- good for wavelength below 320 nm  Alumina Silica Glass- good for lights in visible region  Soft glass cuvettes- preferable for acidic solutions
  21. 21.  converts transmitted light energy into an equivalent amount of electrical energy  Types: ▪ Barrier-layer cell ▪ Photoemissive/phototube ▪ Photomultiplier
  22. 22.  least expensive that is composed of light-sensitive materials such as selenium on iron plate with transparent layer of silver when exposed to light, electrons in the light- sensitive material are excited and are released into the highly conductive silver No need for power source
  23. 23.  similar to barrier-layer cell in that it has photosensitive material that gives off electrons when light energy strikes it composed of positively charged anode and negatively charged cathode enclosed in a glass. The cathode emits electron when exposed to light. The emitted electrons jump over to the positively charged anode where they are collected and return through an external measurable circuit requires an external source of energy
  24. 24.  in this type of photodetector, once the incident light strikes the coated electron, electrons are emitted. The electrons are attracted to a series of anodes known as dynodes. the dynodes gives off secondary electrons once hit by single electrons (initial electron transmission at the cathode triggers a multiple cascade of electrons within the PM tube) the PM tube can amplify the amount of incident light energy therefore it can measure even the very low light levels
  25. 25.  displays the amount of light transmitted
  26. 26.  states that the concentration of a substance is directly proportional to the amount of light absorbed and inversely proportional to the logarithm of the light transmitted
  27. 27.  Percent transmittance (%T)- the ratio of the radiant light transmitted divided by the radiant energy incident to the sample. % Transmittance= (radiant light transmitted / incident light to the sample) x 100
  28. 28.  The sample containing absorbing molecules to be measured is placed in the light path. The difference in amount of light transmitted by the blank and that transmitted by the sample is due only to the presence of the compound being measured.
  29. 29.  Absorbance (A)- is the amount of light absorbed. It can not be measured directly by the spectrophotometer, but rather is mathematically derived from the percent transmittance A = -log (%T) = log 100% - log %T = 2 – log %T
  30. 30.  According to Beer’s law, absorbance is directly proportional to the concentration. A=exbxc  where e- molar absorptivity, b- length of light path, c- concentration  e and b tend to be constant that’s why they can be eliminated
  31. 31.  Au = Cu As Cs Au=Cu, tama? As=Cs, tama? Therefore… how will you compute for the concentration of the unknown?
  32. 32.  In a double-beam spectrophotometer, the light is directed forward in two directions by a rotating chopper. One light path is directed forward the sample cuvette, while, simultaneously the other is directed towards the reference cuvette monochromatic light from single or two identical monochromators pass through both a reference and a sample compartment. The intensity of the two light beams is then measured by one or two photodetectors. The sample beam is then compared to that of the reference beam
  33. 33.  Group 1 metals 1+ charge (Na, K, Li) EXCITABLE……….. EMITS Specific Light
  34. 34.  the measurement of emitted light when electrons in an atom become excited by heat energy produced by the flame. Excited atoms return to ground state by emitting light energy that is characteristic of that atom.
  35. 35.  Na filter- transmits only yellow light (589 nm) K filter - transmits only violet light (367 nm) Lithium – transmits only red light (767nm)
  36. 36.  due to the mixture of hydrogen and oxygen gases (acetylene, propane or natural gas) Purpose of the flame:  breaks the chemical bonds to produce atoms  source of energy that will be absorbed by the atoms to enter the excitation state
  37. 37.  breaks up the solution into finer droplets so that the atom will absorb heat energy from the flame and get excited
  38. 38.  Types of burners: Total consumption burners- the sample is aspirated directly into the flame. The flame can be made hotter but there is production of large droplets in the flame Premix burner- the sample is atomized before entering the flame and does not create noise; gravitational feeding of sample into the flame
  39. 39.  Na filter- transmits only yellow light (589 nm) K filter - transmits only violet light (367 nm) Lithium – transmits only red light (767nm)
  40. 40.  Guess the type of photodetector commonly employed…. Guess the standard employed in FES
  41. 41.  Group two metals 2(+) charges NOT EASILY EXCITED but can ABSORB light!!
  42. 42.  measures concentration of element by detecting absorption of electromagnetic radiation by atoms. The elements are not excited but they are dissociated from their chemical bonds and placed in the unionized, unexcited ground state. -
  43. 43.  Components:  Light Source- hollow cathode lamp,  Mechanical Rotating Chopper- modulates light beam coming from the light source  Burner- uses flame to dissociate the chemical bonds and form free unexcited atoms  Monochromator- selects the desired wavelength from a spectrum of wavelength  Detector  Read-Out Device
  44. 44.  Components:  Light Source- hollow cathode lamp,  Mechanical Rotating Chopper- modulates light beam coming from the light source  Burner- uses flame to dissociate the chemical bonds and form free unexcited atoms  Monochromator- selects the desired wavelength from a spectrum of wavelength  Detector  Read-Out Device
  45. 45.  Interferences:  Chemical – situation at which the flame could not dissociate the sample into neutral atoms  Ionization – situation at which atoms in the flame become excited and emits energy
  46. 46.  Define plssssssssssss….  Fluorometry  Chemiluminescence  Nephelometry  Turbidimetry
  47. 47.  measures the fluorescence or the energy emission that occurs when a certain compound absorb electromagnetic radiation, become excited and then return to an energy state that is usually higher than their original level. -Since the energy given off is less than that of the absorbed, the wavelength of the light given off is usually is longer than that absorbed.
  48. 48.  Emitted light has longer wavelength than the incident/excited light due to the loss of energy during collision, transfer to other molecules, heat loss and emission of radiant energy
  49. 49.  Components:  Light source- Mercury Vapor lamp for high light intensity  Excitation/Primary Monochromator-  Cuvette  Emission/Secondary Monochromator-position at a right angle from the cuvette to eliminate potential interference from the excitation light  Photodetector- probably PMT
  50. 50.  Main Problem with Fluorescence: Quenching- quick disappearance of fluorescence
  51. 51.  the excitation of the substance does not involve electromagnetic radiation and no monochromators are needed, instead the excitation energy comes from a chemical or electrochemical reaction.
  52. 52.  the measurement of the reduction in light transmission caused by particle formation -the amount of light absorbed by the particles depends on the specimen concentration and on the particle size -sample handling can become critical because particles tend to aggregate and settle down.
  53. 53.  useful method to determine the concentration of solutions that contains particles too large for absorption spectrometry
  54. 54.  3 types of scattered lights based on the relative size of the light wavelength:  If the wavelength is larger than the diameter of the particles, the light scatter is symmetric around the particle  If the wavelength is smaller than the diameter of the particles, the light scatters backwards  If the wavelength is approximately the same as the diameter, there will be more forward light scatter
  55. 55.  is the movement of electrically charged compounds in a medium resulting to their separations based on their electrical charges when an electric current is applied -ions move toward opposite electrodes. Cations move towards cathode while anions move toward the anodes.
  56. 56.  is basically an absorbance measurement. A densitometer measures the absorbance of the stain on a support. basic component includes light source, monochromator, movable carriage, photodetector and read-out device
  57. 57.  measurement of the osmolality of an aqueous solution such as serum, plasma and urine; measurement of the concentration of dissolve solute particles in a solution Osmolality of a solution is related to its colligative properties such as osmotic pressure, boiling point, freezing point, and vapor pressure.
  58. 58.  1. Know how to check for the following:  Wavelength accuracy  Stray light  Photometric Accuracy