Techiniques are based off of the measurement of light which is an electromagneteic radiation.
Is photons of energy traveling in waves. Thus light exhibits both wave and particle like properties.
The wavelength of light measured in nm is the distance between adjacent troughs or peaks in a continuous wave.
Properties of light
- E = Energy of photons (h)(v)
- v = (c)/(λ)
- E = (h)(c)/(λ)
Wavelength of light is inversely proportional to the frequency and thus to the energy.
A = (ε)(b)(c)
It is the realtionship between absorption of light by a solution and concentration of that sln.
Also states that the concentration is directly proportional to the amount of light absorbed.
How is the "ε" defined in the beers law
ε is defined as a specific solute in a specific solvent, and a specific monochromatic wavelength of light.
Is the fraction of a specific wavelength of light absorbed by a given type of molecule under prescribed conditions of temp, pH, etc...
If light energy is NOT absorbed it is what?
Reflected or Transmitted
Spectrophotometry measurement are based on?
The detection and quantitation of energy that is transmitted after passing a beam of light throught the sln being analyzed.
Transmitted energy is:
Expressed in terms of % transmittance, which is the ratio of the light transmitted / the light incident on the sample.
- % transmittance = T/I0 x 100
% transmittance can be expressed as absorbance by:
Absorbance is directly proportional to concentration.
This formula can be used as long as Beer's law is followed.
- Concentration unknown = CUNK
- CUNK = (AUNK/ASTD) x CSTD
Is a system to isolate radiant energy of a desired wavelength and exclude that of other wavelengths.
- Entrance Slit
- Wavelength dispersing device
- - Filters
- - Prisms
- - Diffraction gratings
- Exit slit
Is described as photons of energy traveling in waves.
The relationship between wavelength and Energy is:Planks formula
E = hv
Produce polychromatic light:
- Tungsten - visible region 350-950 nm
- Quartz - Extended visible light source
- Vapor Lamps - UV light (deuterium & mercury)
- Lasers - Intense bam of light
The distance between one peak or crest of a wave of light, heat, or other energy and the next corresponding peak or crest.
Spectral bandpass - Monochromators
Width (in nm) of the spectral transmittance curve at a point equal to one-half of the peak transmittance.
Is the range of wavelengths transmitted by a monochromator and measured at 1/2 maximum transmittance.
Readings between cuvettes
current produced in the absence of light.
If concentration of analyte = constant, but...
The light path is increased, the absorbance would also increase therefore, the calculated concentration = inaccuracy
Refers t any wavelength outside the bandtransmitted by the monochromator.
Causes a decrease in absorbance.
Reaseons: ie. scratches, dust particles. etc...
Factors that affect Beer's law
- Concentration of analyte (hight)
- Stray light
- Solvent absorbance
- reation with another substance
contains pos and neg (anode/cathode)
Cathode emits electrons when exposed to light. Collected by anode & returned by cathode.
Double beam in space
One light source split into two beams
Double beam in Time
Light beam split by chopper. Alternately directed to sample then to reference.
This check is different than verification of the linear range of an analyte which requires the analysis of standard sln's over a range of concentration of the analyte.