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167 Cards in this Set
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Transmitted via electromagnetic waves that are characterized by frequency and wavelength |
Energy |
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The number of vibrations of wave motions per second |
Frequency |
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Important in instrumentation; The distance between two successive peaks and expressed in terms of nanometer |
Wavelength |
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Types of wavelengths |
Visible spectrum - 400-700nm Ultraviolet region - <400 nm Infrared region - >700nm |
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Wavelength indicated on the control dial is the actual wavelength of the light passed by the monochromator |
Wavelength accuracy |
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Used to check wavelength accuracy |
Didymium or Holmium oxide filter |
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Verify absorbance accuracy on linearity |
Neutral density filters and dichromate solution |
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PRINCIPLE OF SPECTROPHOTOMETRY |
Measurement of light transmitted by a solution to determine the concentration of light absorbing substances in the solution |
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Spectrophotometry is base on what law? |
Beer Lambert's law where the concentration of a substance is directly proportional to the amount of light absorbed (Absorbance or Optical density) and inversely proportional to the amount of transmitted light (%transmittance) |
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It measures light in specific wavelength |
Spectrophotometer |
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Chemical reaction produces a substance that absorbs light |
Spectrophotomer |
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Absorbance |
It is the amount of light absorbed |
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It is proportional to the inverse log of transmittance It is mathematically derived from %T |
Absorbance |
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Formula of absorbance |
A=abc Where: A = absorbance a = molar absorptivity, absorptivity of the compound under standard conditions b = length of light through the solution c = concentration of absorbing molecules/solution A=2-log%T |
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Formula for unknown solution |
(Au/As) * Cs |
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It is the ratio of the radiant energy transmitted (T) divided by the radiant energy incident on the sample |
Percent transmittance |
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%T= |
(lt/Io) * 100 Where: It = transmitted light thru the sample Io = intensity of light striking the sample |
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In the actual practice, the light transmitted by a ________ is substituted for ____. So the formula is %T= |
Blank, Io, (sample beam signal/ blank beam signal) x 100 |
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Components of Spectrophotmeter |
1. Exciter lamp 2. Entrance slit 3. Monochromator 4. Exit slit 5. Cuvette/Cuvet/ analytical cell/sample cell 6. Photodetector 7. Meter or read-out device |
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Serves as a source of light |
Exciter lamp or light source (ranging from visible to ultraviolet) |
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Two examples of exciter lamp |
1. Tungsten lamp - commonly for VIS and IR 2. Deuterium lamp - routinely used for UV |
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Controls the amount of light that enters the monochromator |
Entrance slit |
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Produces light of specific wavelength from the exciter lamp |
Monochromator |
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Types of monochromator |
1. Prisms 2. Diffraction grating 3. Interference filters |
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Are edging glass, quarts or sodium chloride that disperse light to different angles of refraction due to diff. wavelength |
Prisms |
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Refract and diffract light into diff. spectra. They are made up of grooves (for refraction) and slits (for diffraction) ina an aluminim surface of a flat piece |
Diffraction grating |
Most commonly used monochromator. It has better resolution than prisms |
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Composed of semitransparent silver films on both sides of a dielectric such as magnesium fluoride. They filter and allow transmission of 40 to 60% of incident light with a bandpass or bandwidth between 10 to 20 nm |
Interference filters |
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Controls the width of light beam, allows only a narrow fraction of spectrum to reach the sample cuvet |
Exit slit |
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Holds the sample to be analyzed |
Cuvette/cuvet/analytical cell/sample cell |
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Kinds of cuvet |
1. Alumina silica glass - most commonly used (350-2000nm) 2. Quartz/plastic - solution requiring visible and UV spectra 3. Borosilicate glass - alkaline solution 4. Soft glass - acidic solution |
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Light in frost glass results |
Falsely decrease result |
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Scratch in cuvet can cause |
Light scattering |
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Different substances requires different _____ |
Wavelength |
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Detects and collects the light energy transmitted and converts it into a current to produce a readable output |
Photodetector |
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Types of photodetector |
1. Barrier layer cell 2. Phototube 3. Photomultiplier tubes 4. Semiconductor detectors |
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Have selenium coated with silver serve as a negative electrode and iron base serve as a positive electrode Simplest, least expensive, and temperature sensitive |
Barrier layer cell |
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Most widely used photodetector. It has curved sheet of photosensitive material that serves as the cathode and a positively charged thin tube serves as the anode. Photodetector for single beam |
Phototube |
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Have cathode, anode and electron-multiplying dynodes. It is the photodetector for double beam |
Photomultiplier tubes |
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Including photoresistor, photodiode and phototransistor have replaced conventional phototubes in modern laboratory instrument |
Semiconductor detectors |
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Displays output on the detection system |
Meter or read out device |
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PRINCIPLE OF DOUBLE BEAM SPECTROPHOTOMETER |
Absorbance is measured at two different wavelength using a light from either single or two monochromators that would analyze sample and reference cuvette. |
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Two types of double beam spectrophotometer |
1. Double beam in space 2. Double beam in time |
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PRINCIPLE OF FLAME EMISSION PHOTOMETRY |
It measures the amount of light produced or emmited from excitation of electrons sample by heat energy (flame) |
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Flame emission photomotery usage |
Used: excited ions (Na and K) |
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Components of flame photometers |
1. Gas 2. Atomizer/ burner 3. Entrance slit 4. Monochromator 5. Exit slit 6. Detector |
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Source of flame energy Mixture of hydrogen and oxygen gas, natural gas, acetylene and propane in conjunction with air or oxygen |
Gas (hydrogen, oxygen gas, natural gas, acetylene and propane) |
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Introduces heat energy to atoms and become excited |
Atomizer or burner |
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Used to correct fluctuations |
Reference cell |
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Sample can be |
Serum or plasma |
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Detectors: Lithium - ________ Sodium - ________ Magnesium - ___________ Rubidium - _________ Potassium - __________ And ordinary flame |
Red, yellow, blue, red and violet |
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Is limited to elements that are easily excited by flame |
Atomic emission |
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Internals standards for sodium and potassium measurements |
Lithium and Cesium |
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Internal standards for lithium measurements |
Potassium and cesium |
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Principle of AAS or Atomic Absorption Spectrophotometry |
It measures the amount of light absorb by the element sample in an unexcited state, un-ionized state neutral atom ground state and dissociated from its chemical bond |
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AAS usage |
Unexcited trace metals (Calcium and magnesium) |
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Components of AAS |
1. Hollow cathode lamp 2. Nebulizer/atomizer 3. Monochromator slits 4. Detector |
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Source of radiation energy; Lamp only used in AAS |
Hollow cathode lamp |
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Sprays the sample into the sample |
Nebulizer/atomizer |
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Isolates light by filtering out extraneous energy from the flame |
Monochromator slits |
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Interference in AAS |
1. Chemical interference 2. Ionization interference 3. Matrix interference |
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Occurs when flame cannot dissociate the sample |
Chemical interference |
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Occurs when samples in flame become excited |
Ionization interference |
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-Occurs when light absorption is enhanced by organic substance -Occurs when sample is evaporated in the flame and produce a solid particle |
Matrix interference |
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Added to samples to form a stable complexes with phosphate |
Lanthanum or Strontoum Chloride |
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Photodetector sa AAS |
Photomultiplier |
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Volumetric is also known as |
Titrimetry |
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PRINCIPLE OF VOLUMETRIC |
The unkown sample is made to react with a known solution in the presence of an indicator |
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Examples of volumetric |
Schales and Schales method (chloride test) EDTA titration method (Calcium test) |
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Fluorometry is also known as |
Molecular Luminescence Spectroscopy |
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Only one that use two monochromator |
Primary monochromator - for excitation Secondary monochromator - for emission |
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AAS AND FEP is replaced by |
Ion-selective electrode |
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PRINCIPLE OF FLUOROMETRY |
It measures the amount of light emitted by a substance due to its excitation from a source rendering a higher or equal energy from its original state |
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Fluorometry usage |
Use: Porphyrins, magnesium, calcium and cathecolamines
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The wavelength of emitted light is longer than the excited light It uses electromagnetic radiation |
Fluorometry |
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Types of luminescence |
1. Fluorescence 2. Phosphorescence 3. Chemiluminescence |
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Components of fluorometer |
1. Light source 2. Excitation monochromator (primary) 3. Cuvette 4. Emission monochromator (secondary) 5. Photodetector |
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PRINCIPLE OF NEPHELOMETRY |
It measures the amount of light scattered by a particular solution. Light scattering depends on the particle size and light wavelength |
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Nephelometry usage |
Use: Antigen-antibody complexes (proteins) |
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Three types of light scatter based on the size of the light wavelength and diameter of the particle |
Large size of light wavelength - small diameter of the particle, the light scatter is symmetric around the particle
Small size of light wavelength - large diameter of the particle, the scatter backward (back scatter)
Approximately the same - the light scatter in forward direction (forward scatter) |
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The light scattered is measured at _______ of the cuvette |
15-90 degrees |
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Components of Nephelometer |
1. Light source includes: - Mercury-arc lamp - Tungsten filament lamp - Light-emmited diode - Laser produce stable, monochromatic light of narrow bandwidth. It emits radiant energy that is coherent, parallel and polarized. It is more sensitive than conventional instruments because of its high intensity monochromatic beam 2. Collimator 3. Monochromator 4. Cuvette 5. Stray light trap 6. Photodetectors |
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Is a device that produce a beam of parallel rays |
Collimator |
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PRINCIPLE OF TURBIDIMETRY |
It measures the amount of light reduced or blocked by particle formation |
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Application of Turbidimetry |
1. Detection of bacterial growth and bacterial culture 2. Antibiotic sensitivity 3. Coagulation studies 4. Protein concentration in CSF and urine |
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Specimen concentration is directly proportional to the amount of light blocked |
Turbidimetry |
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PRINCIPLE OF REFRACTOMETRY |
It measures the amount of light bent or deflected in a straight path of a particular substance |
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The refractivity of solution is an indirect measurement of total solute concentration |
Refractometry |
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Application of refractometry |
Total serum protein concentration Specific gravity of urine Column effluent of HPLC |
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PRINCIPLE OF OSMOMETRY |
It measure the amount of dissolved substance in a solution (osmolality) |
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Most osmometers (micro osmette and osmette II) use ______ as a reference to determine osmolality |
Freezing point |
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Colligative properties |
Osmotic pressure, boiling point, freezing point and vapor pressure |
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PRINCIPLE OF DENSITOMETRY |
It measures the amount of light absorbed |
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Components of densitometer |
Light source Monochromator Scanner Optical system Photodetector
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Application of densitometer |
Usually used used in electrophoresis, where the concentration is directly proportional to the stained molecule which is measured |
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PRINCIPLE OF FLOW CYTOMETRY |
It measures the amount and properties of cells suspended in a moving liquid medium |
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In flow cytometry, cells are distinguished by _________ and _________ |
-Forward scattering light for cells size, and 90 scattering for cell granularity - Fluorescent light for cell population |
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Application of flow cytometry |
- Analysis of number of cells, cell size and surface and cytoplasmic markers - Immunophenotyphing for leukemia and monitoring of immune status - Cell cycle analysis by using propidium iodide that binds DNA and quantifying cells in different stages of cell cycle - CBC for erythrocytes, platelets, and leukocytes along with five-part differential for leukocytes |
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PRINCIPLE OF ELECTRICAL IMPEDANCE |
It is based on change of electrical resistance of an aperture as particle passes through |
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Application in electrical impedance |
used in hematology measuring erythrocytes, platelets and leukocytes |
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Machines involved in electrical impedance |
Coulter counter Abbot Cell Dyn |
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Uses electric energy Measures current or voltage |
ELECTROCHEMISTRY TECHNIQUES |
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Measurement of differences in voltage (potential) at a constant current |
Potentiometry |
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Measurement of electrical potential due to the activity of free ions - change in voltage indicates activity of each analyte |
Potentiometry |
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Reference electrodes in potentiometry |
Calomel (Mercuric chloride) and silver-silver chloride (pH) |
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Example of potentiometry |
pH and pCO2 test |
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An electrochemical transducer capable of responding to one given ion |
Ion selective electrode (ISE) |
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Convert specific activity of ion into a electric current |
Transducer or sensor |
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Its ionic selectivity depends on the membrane/barrier composition used |
ISE |
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Measure the electrolyte dissolved in the fluid phase of the sample in mmol/L of plasma water |
ISE analyzers |
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2 types of ISE |
1. Direct ISE - w/o sample dilution; most commonly used 2. Indirect ISE - w/ sample dilution |
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ISE MEMBRANE for Sodium |
Glass aluminum silicate |
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ISE MEMBRANE for Potassium |
Valinomycin gel |
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ISE MEMBRANE for Calcium and Lithium |
Organic liquid membrane ion exchanges |
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ISE MEMBRANE for Chloride |
Tri-n-octylpropylammonium decanol chloride |
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Measures the hydrogen activity Consists of a small bulb made of layers of hydrated and non-hydrated glass which contains a chloride ion buffer solution |
pH electrode |
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pH |
H+ electrodes |
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pCO2 |
Severing haus electrode |
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pO2 |
Clark |
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Cl |
Cotlove Chloridometer - sweat chloride |
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Potentiometry follows the |
Nernst equation |
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The voltage is theoretically dependent to the specific activity of ions to be analyzed |
Nernst equation |
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Measurement of the amount of electricity (in coulombs) at a fixed potential |
Coulometry |
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Electrochemical titration in which the titrant is electrochemically generated and the endpoint is detected by amperometry |
Coulometry |
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Example of coulometry |
Chloride test (serum and sweat, CSF) |
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Interferences in Coulometry |
Bromide Cyanide Cysteine |
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Coulometry follows the |
Faraday's law |
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Measurementt of the current flow produced by an oxidation-reduction |
Amperometry |
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Examples of amperometry |
pO2, glucose, chloride and peroxidase determinations |
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Measurement of difference in current at a constant voltage |
Polarography |
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Polarography follows the |
Ilkovic equation |
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- Measurement of current after which a potential is applied to an electrochemical cell - it allows sample to be preconcentrated, thus utilizing minimal analyte |
Voltammetry |
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Lead and Iron testing |
Anodic stripping voltammetry |
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Migration of charged particles in an electic field |
Electrophoresis |
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Separates protein on the basis of their electric charge densities |
Electrophoresis |
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Factors affecting rate of migration |
Net electric charge of the molecule Size and shape of the molecule Electric field strength Nature of the supporting medium Temperature of operations |
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Supporting media |
Paper electrophoresis Starch gel Cellulose acetate Agarose gel Polyacrylamide gel |
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Separates by surface charge and molecular size |
Starch gel |
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Separates by molecular size |
Cellulose acetate |
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Neutral, spearates by electric charge |
Agarose gel |
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Neutral, separates on the bands of charge and molecular size; separates proteins into 20 fraction; used to study isoenzymes |
Polyacrylamide gel |
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Substances with same catalytic activity but slightly diff. molecular structure |
Isoenzymes |
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Isoenzymes |
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Involves the separation of soluble components in a solution by specific difference in physical-chemical characteristics of the different constituents |
Chromatography |
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Two forms of chromatography |
Planar Column |
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- Most simple - Fractionation of sugar and amino acids |
Paper chromatography |
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It is used for drug screening (semiquantitative screening test) |
Thin layer chromatography |
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Relative distance of migration from the point of application |
Retention factor |
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Basic component of Chromatography |
Mobile phase Stationary Column holding the stationary phase Separated components |
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Rf= |
Distance leading edge of component moves/total distance solvent front moves |
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Used for separation of steroids, barbiturates, blood, alcohol and liquids |
Gas chromatography |
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Two types of GC |
Gas solid chromatography Gas liquid chromatography |
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Differences in absorption at the solid phase surfaces |
Gas solid chromatography |
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Separation occurs by difference in solute partitioning between the gaseous mobile phase and the liquid stationary phase |
Gas liquid chromatography |
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Based on the fragmentation and ionization of molecules using a suitable source of energy |
Mass Spectrometry |
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Gold standard for drug testing |
Gas Chromatography Mass spectroscopy |
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Can detect 20/20 inborn errors of metabolism from a single blood spot |
Tandem mass spectroscopy (MS/MS) |
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Based on the distribution of solutes between a liquid mobile phase and a stationary phase |
Liquid Chromatography |
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Most widely used LC |
HPLC |
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- Used for fractionation of drugs, hormones, liquids, carbo and proteins - Uses pressure for fast separation |
HPLC |
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For detecting non-volatile substances in body fluids |
LC-MS |
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All samples are loaded at the same time, a single test is conducted on each sample |
Batch testing |
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More than one test is analyzed concurrently on a given clinical specimen |
Parallel testing |
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Any test can be performed on any sample in any sequence |
Random access testing |
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Multiple test analyzed one after another on a given specimen |
Sequential testing |
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A system other than the manufacturer's reagent can be utilized for measurement |
Open reagent system |
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A system where the operator can only used the manufacturer's reagents |
Closed reagent system |
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Three basic approaches to automation |
Continuous flow analyzer Centrifugal analyzer Discrete analyzer |
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