CONCEPTS IN INSTRUMENTATION Flashcards
Methods of determining the concentration of substance in solution by measuring the amount of light absorbed by that solution after appropriate treatment
Spectrophotometry
Visible light falls between:
400 nm - 700 nm
BEER-LAMBERT LAW (BEER’S LAW) states that:
The concentration of a substance is directly proportional to the logarithm of the transmitted light
In the Beer’s formula A = abc, the “b” represents:
b is the length of light path through the solution
In the Beer’s formula A = abc, the “a” represents:
a = Molar absorptivity, the fraction of a specific wavelength of light absorbed by a given type of molecule.
Components of a spectrophotometer:
- Light source
- Monochromators
- Sample cell/cuvet
- Photodetectors
Most common source of light for work in the visible and near-infrared regions:
Incandescent tungsten or
Tungsten-iodide lamp
Most commonly used for ultraviolet (UV) work:
Deuterium - discharge lamp &
Mercury - arc lamp
Most commonly used Monochromator (isolate desired wavelength):
Diffraction gratings
Preferred sample cell; advantage over round cuvets in that there is less error from the lens effect, orientation in the spectrophotometer, and refraction
Square sample cell
Sample cell used for applications in the visible rage
Glass cuvet
Sample cell for applications requiring UV radiation:
Quartz cuvet
Used in instruments designed to be extremely sensitive to very low light levels and light flashes of very short duration
Photomultiplier (PM) tube
Measures the quantity of light reflected by a liquid sample that has been dispensed onto a grainy or fibrous solid support
Reflectometry
Application of Reflectometry:
- Urine dipstick analysis
- Dry slide chemical analysis
Measurement of concentration is done by detecting the absorption of electromagnetic by atoms rather than molecules.
Atomic Absorption Spectrophotometry (AAS)
Components of AAS:
Hollow-cathode lamp - usual light source
Flame - breaks chemical bonds and form free, unexcited atoms; serves as sample cells (instead of a cuvet)
Monochromator
Applications of AAS:
measurement of unexcited trace metals e.g. calcium and magnesium
*Reference method for Ca and Mg
Measurement if light emitted by excited atoms (flame excites atoms)
Flame photometry
Some metals produce characteristic flame colors:
Lithium:
Sodium:
Magnesium:
Rubidium:
Potassium:
Some metals produce characteristic flame colors:
Lithium: RED
Sodium: YELLOW
Magnesium: BLUE
Rubidium: RED
Potassium: VIOLET
Applications of Flame photometry:
Widely used before to determine the concentration of Na, K, Li
*Now: ISE (Ion-Selective Electrode) for Na, K, Li
Measurement of the concentration of solutions that contain fluorescing molecules:
Fluorometry
Most common light source of fluorometry:
Xenon lamp
How many monochromators does fluorometry have?
2 monochromators set up at 90 degree angle
Applications of Fluorometry:
Fluorometry us used to measure small particles, such as drugs.
Chemical energy generated in a chemiluminiscent reaction produces excited intermediates that decay to a ground state with the emission of photons; no excitation is required unlike fluorometry:
Chemiluminescence
Measurements are made with a spectrophotometer to determine concentration of particulate matter. The amount of light blocked by a suspension of particles depends not only on concentration but also in size
Turbidimetry
Applications of turbidimetry:
- Detection of bacterial growth and bacterial culture
- Antibiotic sensitivity
- Coagulation studies
- Protein concentration in CSF and urine
Light scattered by small particles is measured at an angle to the beam incident to the cuvet.
Nephelometry
Involves measurement of the current or voltage generated by the activity of specific ions. Analytic techniques include potentiometry, coulometry, voltametry, and amperometry`
Electrochemistry
Measurement of potential (voltage) between two electrodes in a solution to measure analyte concentration
Applications:
pH, pCO2, Na, Ca, K, NH4, electrolytes
Potentiometry
Measurement of the current flow produced by an oxidation-reduction reaction
Application:
pO2 (Clark electrode), glucose, peroxidase
Amperometry
Electrochemical titration in which the titrant is electrochemically generated
Applications:
Cl
Coulometry
Potential is applied to an electrochemical cell and the resulting current is measured
Application: Anodic stripping voltametry (for lead and iron)
Voltametry
Separation of charged compounds based on their electrical charge
Electrophoresis
Maintains the pH in electrophoresis:
Buffer
Detecting system of electrophoresis:
Densitometry
Movement of buffer ions and solvent related to the fixed support
Electroendosmosis
Most common and reliable way for quantification of separated protein fractions
Densitometry
Separation of complex mixtures on the basis of different physical attractions between the individual compounds and the stationary phase of the system
Chromatography
Components of chromatography:
- Mobile phase (gas or liquid): carries the complex mixture
- Stationary phase (solid or liquid) substance through the mobile phase flows
- Column: holds the stationary phase
- Eluate: separated components
Chromatographic procedure that uses pressure for faster separations:
High-Performance Liquid Chromatography (HPLC)
Chromatographic procedure that separates mixtures of compounds that are volatile or can be made volatile:
Gas Chromatography (GC)
Sample in a MS is first volatilized and then ionized to form charged molecular ions and fragments that are separated according to their mass-to-charge (m/z) ratio
Mass Spectrometry
Gold standard for drug testing when coupled with GC:
GC/MS
What does LASER stand for:
L = Light
A = Amplification by
S = Stimulated
E = Emission of
R = Radiation
