Spectro, Electro Flashcards
Provide foundation for all the measurements in the modern clinical chemistry
Analytical Techniques and Instrumentation
Measurement of light transmitted by a solution in a much narrower wavelength to determine the concentration of light absorbing substances in the solution
Spectrophotometer
It is transmitted via electromagnetic waves and characterized by their frequency and wavelength
Energy
It is the distance between two successive peaks, expressed in terms of nanometer
Wavelength
Number of vibrations of of wave per second
Frequency
A form of electromagnetic radiation consisting energy traveling in waves.
Light
Electromagnetic radiation consist of:
Gamma rays, x-rays, ultraviolet, visible, infrared, microwaves and radiowaves
States that the concentration of a substance is directly proportional or inversely proportional
Beer’ law
Absorbance
A=abc= 2-log%T
a= molar absorptivity
b= length of light through the solution
c= concentration of absorbing molecules
Transmittance
%T= It/ Io x 100
It= transmitted light through the sample
It= transmitted light striking the sample
Components of spectrophotometer
Light source
Entrance slit
Monochromator
Exit slit
Cuvet
Photodetector
Provides polychromatic device /radiant energy to measure the analyte of interest
Light source
Emits radiation that changes in intensity
Continuum source
Examples of contiuum source:
Tungsten- used light source in the visible
Deuterium- used to provide UV radiation
Xenon- discharge lamp, covers both the UV and visible range.
Emits limited radiation and wavelengths
Line source
Examples of line source
Mercury and sodium vapor lamps- uv and visible region
Hollow and cathode lamp- for atomic absorption spectrophotometers
Minimizes stray light or unwanted light and prevents the entrance of scattered light
Entrance slit
Isolate specific wavelengths of light
Monochromator
Types of monochromator
Prism
Diffraction gratings
Wedge-shaped pieces of glass or quartz, allows transmission of light, allowing only the desired wavelength to pass through an exit slit
Prism
Small grooves cut, behave like a very small prism, separates white light into various color
Diffraction gratings
Simple, least expensive
Produce monochromatic light based on the principle of constructive interference of waves
Filters
Controls the width of light beam
Exit slit
Used to hold the solution
Cuvette
Types of cuvette
Borosilicate glass
Quartz or plastic
Alumina silica glass
Can convert transmitted energy into equivalent amount of electrical
Photodetector
Types of photodetector
Photocell
Photoemissive
Photomultiplier tube
Amount of light passing through the sample is indicated by a moving needle on a dial or digital display.
Readout device
Migration of charged particles in an electric field
Electrophoresis
What are the charge of proteins during electrophoresis
Negative
Components of electrophoresis
Electrical power, support medium, buffer,sample and detecting system
Factors affecting migration
Net electric charge of the molecule
Size and shape of molecule
Electric field strength
Nature of supporting medium
Temperature of operation
Its function is to supply electrical power by providing constant current,voltage and power
Power supply/ electrical power
Carries the applied current, establishes the pH at which electrophoresis is performed, determines the electrical charge on the solute
Buffers
Effects of the buffer’s ionic strength
Conductance of the support
The thickness of the ionic cloud surrounding a charged molecule
Provides the matrix in which the separation takes place
Supporting medium
Earliest support medium
Paper
Used to separate macromolecules on the basis of both surface charge and molecular size
Starch gel and cellulose acetate
Supporting media of choice for electrophoresis, separation is based on charge to mass ratio of the protein
Agarose
It is thermostable, transparent, strong and relatively chemically inert
Polyarcylamide gel
It holds the buffer solution and the supporting medium
Electrophoresis chamber
