chem unit 2 chapter 4

Instrumentation, Lab Automation and Informatics – Part 1

• PowerPoints are for lecture notes only, the textbook is required for the exam
• Unit Objectives from the textbook are the study guide, not the PowerPoint
• Test questions are based on the UNIT OBJECTIVES from the textbook
• Absorption spectroscopy is a technique to measure radiation absorption as it interacts with a sample.
• Absorption spectrum shows the frequencies of light transmitted, with dark bands where electrons absorb energy.
• The sample interacts with electromagnetic radiation (EMR) in the form of photons from the radiating field.
• Absorption intensity varies with frequency creating the absorption spectrum.
• The interaction of EMR with matter is the principal method of measuring biological fluid analytes.
• The measuring device (black box) remains fundamentally the same despite analytical system changes.
• Radiation transmission in matter can be viewed as momentary radiant energy retention.
• The radiation is then re-emitted in all directions as particles return to their original electronic state.
• Rayleigh scattering occurs when particles are significantly smaller than the wavelength of radiation (e.g., blue sky).
• Tyndall effect is scattering visible by colloidal particles.
• Raman scattering involves photon absorption and vibrational excitation energy variations.
• Spectrophotometric techniques use ultraviolet or visible light.
• Lambert's law describes the decrease in radiant power as light path increases arithmetically, for constant concentration systems.
• Beer's law describes the linear relationship between absorbance and concentration for monochromatic radiation.
• Absorbance is proportional to light path (b) and concentration (c) of the absorbing species.
• A typical photometer or spectrophotometer has five main components (single or double beam): stable radiant energy source, device isolating a specific region of the electromagnetic spectrum, sample holder, photo detector, and read-out device.
• Radiant sources are either continuum (intensity varies slowly wrt wavelength) or line (discrete lines/bands).
• Wavelength selectors (Monochromators):
  • Modified spectroscope for selective transmission of a narrow band of a spectrum.
  • Quality is measured in nominal wavelength, effective bandwidth and bandpass for specific transmittance of the wavelength band.
  • Absorption filters and interference filters.
  • Prism and grating monochromators.
• Cuvettes hold samples of material that is transparent to radiation in the pertinent spectral region.
• Radiation transducers translate energy types (e.g., photovoltaic cell, barrier layer cell, vacuum phototube, photomultiplier tubes, silicon diode transducers, multichannel photon transducers).
• Photomultiplier tubes (PMTs) amplify signals when radiant power is low.
• Signal processors process transducer signals (amplification, rectification, altering signal phase and filtering unwanted components).
• Optimal photometric performance needs periodic monitoring of wavelength accuracy, bandwidth, photometric accuracy, linearity and stray light. Methods use known absorbances of glass filters/solutions at specific wavelengths.
• Spectroscope, colorimeter, and photometer are different types of photometric instruments.
• Spectrophotometers or spectrometers measure radiation intensity as a function of wavelength/frequency, with exit slits and photoelectron transducers to determine beam ratio.
• Double beam spectrometers (space/time designs) split/chop the beam of radiation for measurements.
• Reflectometers are filter photometers that determine light reflected from a nonpolished sample surface (specular/diffuse).
• Tungsten-quartz halide lamp sources are often available in reflectometers.
• Light passes through slits to a surface (e.g., dry slide/urine dipstick) with a wavelength selector or filter wheel to isolate the desired wavelength.
• Solid-state photodiodes measure the reflected radiant energy.
• Atomic Absorption Spectroscopy (AAS) uses absorption of EMR to measure metals like calcium, lead, copper and lithium concentrations in solution. Its labor intensive and time-consuming work requires precise lab techniques. The amount of absorbed radiation is proportional to the metal's concentration in solution in terms of G°.
• Molecular Luminescence Spectroscopy uses compounds' unique excitation and emission wavelengths for high sensitivity and specificity. Fluorescent and phosphorescence light is emitted from excitation of singlet/triplet states; differentiating these states is easily achieved.
• Fluorometers use detectors at 90° to polychromatic light sources. Types of transducers, such as PMT are used. Typical cuvette/cell shapes are often rectangular or cylindrical and can be made of glass/quartz.
• Fluorometry and other applications use fluorescence polarization, time-resolved assays and front-surface fluorometry.
• Chemiluminescence is the light production through chemical/electrochemical reactions instead of EMR.
• Nephelometry measures scattered light. A typical setup includes a light source, collimator, monochromator, sample cuvette and detectors.
• Turbidimetry measures reduced light transmission due to particle formation in solutions. It detects forward direction light scattering. The amount of scattered light is related to the specimen particle concentration and size.
• Refractometry measures light refraction from a medium (e.g., glass, water) reflecting light speed differences across boundaries. Key is critical angle and refractivity.
• Osmometry measures the osmolality of aqueous solutions (serum, plasma, urine). Osmotic pressure increases with increasing osmolality (boiling point elevation, freezing point depression and vapor pressure depression). The freezing point osmometer measures the freezing point to determine osmolality.
• Potentiometry measures electrical potentials at metal-solution interfaces (e.g., reference and indicator electrodes). Nernst equation describes cell potential with concentration variations. Often use reference electrode with constant voltage and indicator electrode for measurement.
• Specific ion electrodes such as sodium, lithium, calcium, magnesium, hydrogen, chloride, potassium are used in potentiometry with key membrane components influencing function such as silicate, dodecyl, phosphate, tridodecyl, valinomycin.
• pH electrodes are small bulbs from non/hydrated glass layers with chloride buffer.
• PCO2 electrodes have membrane permeable to CO2.
• Coulometry measures electricity needed to convert analyte to different oxidation states. A coulomb is one ampere of current for one second.
• Amperometry uses current produced by redox (e.g., chloride) reactions with pairs of indicator electrodes. Clark PO2 electrodes have gas-permeable membranes (polypropylene) to measure dissolved oxygen.
• Voltammetry uses current measurements dependent on applied potential (e.g., anodic stripping voltammetry for lead analysis with reduction/deposition, resting, and oxidation phases).
• Conductometry measures solutions’ ability to conduct electricity related to reciprocal resistance (conductance). Also measures resistivity in relation to the size and concentration of a sample (e.g. for blood cells). Impedance measurements also use this principle to differentiate cells.
• Electrophoresis separates charged compounds (proteins) in a solid/semisolid medium through a current. The pH at which an amino acid is a zwitterion is the isoelectric point (pI).
• Densitometry measures absorbance of stained compounds on a medium using a light source, monochromator, and moveable carriage for photodiode detection.
• Capillary Electrophoresis uses fused silica capillaries with electrolyte buffer reservoirs and a high-voltage power supply, detector, and data acquisition unit (e.g., pH gradient applied; useful for protein isoenzyme measurements).
• Chromatography methods such as column chromatography, thin-layer chromatography, gas chromatography, and high-performance liquid chromatography are useful techniques for separating chemical compounds. Retention time (R_t), resolution (R_s) are characteristics of these methods; Column retention factor (k'), column efficiency (N), and column selectivity (α) are useful metrics. Chromatography factors like solvent strength, temperature, flow rate, column length, and particle size all play roles in results.
• Thin-layer chromatography (TLC) uses a stationary phase (silica) bonded to a solid support (glass) for separation.
• Gas chromatography (GC) uses carrier gas through columns to separate thermolabile compounds. Optimized performance depends on accuracy, sensitivity, and reproducibility of detectors, also response time of the detector and temperature range of the machine are useful metrics.
• High-performance liquid chromatography (HPLC) uses high pressure and small particles for separating compounds. Isocratic and gradient methods use single or multiple mobile phases.
• Mass spectrometry identifies compounds, measures concentrations and determines the molecular structure of organic/inorganic material. It uses atomization, ion formation, ion separation (m/z), and ion counting for measurements (electrospray ionization, matrix-assisted laser desorption/ionization, surface-enhanced laser desorption/ionization) .
• Different types of mass analyzers (e.g. time-of-flight, quadrupole ion trap, Fourier transform ion cyclotron mass spectrometers) are often used.
• Scintillation counters detect gamma rays and charged particles using scintillators which generate light. A photomultiplier tube (PMT) detects the light, measures the electrical signal, and counts scintillations.
• Nuclear magnetic resonance spectroscopy (NMR) measures nuclear spin energy state changes when immersed in a static magnetic field and oscillated.
• Flow cytometers measure multiple properties of particles flowing past a light source (multiple detectors measure different angles/properties/characteristics).
• Microscale technologies (µTAS) offer total integration of steps such as sample prep, separation, detection, quantification on a microchip surface (micro machining, optodes). Biosensors are also part of microscale technologies with different transducers (electrochemical, potentiometric, amperometric, conductimetric, piezoelectric, calorimetric, optical - use of biological components and their interaction with a system that translates the information to an electrical signal).
• Point of Care (POC) Testing is any test performed at the time of testing to enable a timely decision to improve health outcome. Devices should be portable, have reagent cartridges, produce results in minutes, and have flexible test options.