CC LEC M3 U1

• Module Objectives:

  • Differentiate the methods of instrumentation as to their principle of operation, components, advantages, limitations, and application in the measurement of different analytes
  • Practice appropriate preventive maintenance on different instruments used in clinical chemistry
• Module Self-Monitoring Form activities include reading module introduction, contents, and objectives, engaging, exploring, explaining, elaborating, and evaluating different topics like spectrometry, luminescence, electro-analytical methods, chromatography, automation, and point of care testing
• Unit 1: Spectrometry:

  • Engage, explore, explain, elaborate, and evaluate the topic
• Unit 2: Luminescence:

  • Engage, explore, explain, elaborate, and evaluate the topic
• Unit 3: Electro-analytical Methods:

  • Engage, explore, explain, elaborate, and evaluate the topic
• Unit 4: Chromatography:

  • Engage, explore, explain, elaborate, and evaluate the topic
• Unit 5: Automation:

  • Engage, explore, explain, elaborate, and evaluate the topic
• Unit 6: Point of Care Testing:

  • Engage, explore, explain, elaborate, and evaluate the topic
• Analytic techniques and instrumentation provide the foundation for all measurements made in a modern clinical chemistry laboratory
• The majority of techniques in analytic chemistry fall into four basic disciplines: spectrometry, luminescence, electroanalytic methods, and chromatography
• Spectrophotometry/Photometry/Colorimetry involves the measurement of radiant energy emitted, transmitted, absorbed, or reflected under controlled conditions
• Spectrophotometry uses a monochromator to select the desired region of the spectrum for measurement, with slits isolating a narrow beam of light to improve its chromatic purity
• Beer's Law explains the relationship between absorbance at a given wavelength and concentration, expressed as A = abc, where A is absorbance, a is absorptivity, b is light path, and c is concentration
• Percent transmittance is the ratio of radiant energy transmitted divided by the radiant energy incident on the sample, with 0% T indicating all light absorbed and 100% T when no light is absorbed
• Absorbance is defined as -log(I/I₀) = log(100%) - log % T = 2 - log % T
• According to Beer’s law, absorbance is directly proportional to concentration
• A spectrophotometer is used to measure the light transmitted by a solution to determine the concentration of the light-absorbing substance in the solution
• Components of a Spectrophotometer:

  • Light Source:
  • Incandescent Lamps:
  • Tungsten/Tungsten Iodide Lamp: emits light in the visible portion of the spectrum, with most emitted in the IR region
  • Hydrogen and deuterium lamps provide continuous spectra in the UV region
  • Low Pressure Mercury Arc Lamp emits a discontinuous or sharp line spectrum with both UV and visible lines
  • Hollow cathode lamp has a very-narrow-wavelength intense source
  • Lasers Sources: used as a light source in spectrophotometers
  • LED (Light Emitting Diodes) made up of semiconductor (aluminum gallium arsenide) and insulator
  • Device for Spectral Isolation achieved by monochromators
• Filters used as monochromators:

  • Glass filter: simple filter made of colored glass
  • Sharp-cutoff filter: eliminates light below a given wavelength
  • Narrow-bandpass filters: constructed by combining sharp cutoff filters or regular filters
• Prisms and Gratings:

  • Prism: separates white light into a continuous spectrum through refraction
  • Diffraction grating: prepared by depositing a thin layer of aluminum-copper alloy on a glass plate, then making small parallel grooves in the metal coating
• Sample Cell:

  • Also known as a cuvet/cuvette or absorption cells
  • Used to hold a liquid sample to be analyzed in the light path of a spectrometer
  • Cuvets may be round, square, or rectangular, with advantages in accuracy and less error from the lens effect
• Quartz cells are used for readings below 340 nm, while plastic cells are designed for disposable, single-use applications
• Cuvets must be clean and optically clear; scratched optical surfaces scatter light and should be discarded
• Cuvets used in the UV region should be free from invisible scratches, fingerprints, or residual traces of previously measured substances which may significantly affect absorbance readings
• Photodetectors convert transmitted radiant energy into electrical energy proportional to the number of photons striking its photosensitive surface
• A photomultiplier (PM) tube is commonly used for measuring light intensity in the UV and visible regions of the spectrum; it has extremely rapid response times, is very sensitive, and has adequate sensitivity over a wide wavelength range
• Solid-state detectors like photodiodes are capable of measuring light at a multitude of wavelengths; when consisting of a two-dimensional array of diodes, each responding to a specific wavelength, it is known as a photodiode array
• Charge-coupled detectors are multi-channel devices with good dynamic ranges and signal to noise ratios superior to those of PM tubes; they are used for molecular fluorescence measurement of very low concentrations of fluorescent molecules
• Photocells, also known as barrier-layer cells, are used in older instruments; they are inexpensive and durable but temperature-sensitive and non-linear at very low and very high levels of illumination
• Phototubes are similar to photocells but require an outside voltage to operate and are more sensitive; they contain an anode and cathode in a glass case
• Readout devices display electrical energy from a detector on a meter or readout system; they can be direct reading or null point systems, with digital readout devices providing visual numerical displays of absorbance or converted values of concentration
• A single beam spectrophotometer is composed of the basic components of a spectrophotometer, deriving a single measurement where the concentration of a solution can be determined
• A double beam spectrophotometer splits light into two beams before reaching the sample; the measurement displayed comes from the ratio of the two beam intensities
• Flame photometry is based on the principle that excited atoms, when returning to the ground state, emit light energy characteristic of the atomic species
• Flame photometers are no longer routinely used in clinical chemistry laboratories due to the development of ion selective electrodes for analytes like Na, K, or Li
• Flame photometry can determine only the concentrations of pure metals; it involves an aspirator, atomizer, flame, lenses, monochromator, and a detector
• The FEP monochromators used for common analytes are: Sodium (589nm) emitting a yellow flame, potassium (767nm) emitting a violet flame, and lithium (671nm) emitting a red flame
• Advantages of flame emission photometry include an accuracy and precision of +/- 1-5% in aqueous solution, suitability for many metallic ions, and being a fast, simple analytical method for determining trace metal ions in solution with relatively few interferences from other elements
• Flame Atomic Emission Spectrophotometry (FEP) is used in routine clinical labs primarily for sodium and potassium measurements
• Lithium levels in the serum are determined by FEP for manic-depression patients on lithium therapy