LIGHT MEASURING SYSTEMS Flashcards
Formula for Absorbance
2 - log%T
What is a bandpass ?
- range of wavelengths that reach the exit slit of a monochromator (max wavelength - min wavelength)
- transmitted at a point equal to half the peak intensity transmitted
Range of wavelengths for UV light or Visible light
UV light: 200 to 300 nm
Visible light: 300 to 700 nm
How do spectrophotometers measure transmittance ?
By comparing the signal generated by the transmitted light (Is) to the signal of the incident light (Io)
The relationship between absorbance and %T is __
The relationship between absorbance and %T is logarithmic
Formula for Absorbance under Beer-lambert’s law
A = εbc
ε = molar absorptivity (L/ mol x cm)
b = light path (cm)
c = concentration of analyte (mol/L)
State the limitations of the Beer-Lambert law
- monochromatic light (Io)
- solvent has insignificant absorbance to analyte
- analyte follows Beer-lambert’s law
- standard and unknown are measured in matched cuvettes
- the concentration range obeys Beer-lambert’s
- absorbance of unknown < absorbance of standard; a dilution can be done
- no optical interferences
- no side reactions between analyte and other molecules
- sides of cuvette are parallel
- no stray light
What are spectral interferences ?
- observed when a compound causes a response similar to that of the analyte
ie. HIL
Explain the principle of reflectance spectrophotometry
- polychromatic light is directed to a diffraction grating or light-emitting diodes to produce monochromatic light
- monochromatic light hits the test surface, light is absorbed and reflected
- color intensity increases with [analyte], absorption increases, and diffuse reflection decreases
- a single wavelength of the diffuse reflected light is selected and directed onto a photodetector
Define reflection density (DR)
A measure of the amount of light absorbed by the chromophore on the test surface:
DR = log (R0 / Rtest)
where,
R0 = light reflected by the white standard
Rtest = light reflected by the surface
In reflectance spectrometry, the relationship between analyte concentration and reflectance is not __, it is __.
In reflectance spectrometry, the relationship between analyte concentration and reflectance is not linear, it is logarithmic
What is Bichromatic Analysis ?
- used to correct for spectral interferences when the MAX absorbance of the analyte = MAX absorbance of interference
- two readings are made: one at MAX absorbance (A1) and one where analyte minimally absorbs light (A2)
- the standard curve is based on A1 = A2 or the ratio (A1/A2)
The Beer-lambert law states that the __ of a substance is __ to the amount of radiant energy __
The Beer-lambert law states that the concentration of a substance is directly proportional to the amount of radiant energy absorbed
What is Allen Correction ?
- multi-chromatic analysis of a rxn to correct for background absorbance
- two wavelengths, in addition to the MAX absorbance are monitored to subtract average background absorbance
ie. A(510nm) = 0.100, A(520) = 0.800, and A(530nm) = 0.300
Corrected A = A520 - [(A510 + A530) / 2]
The Beer-lambert law states that the __ of a substance is __ to the amount of radiant energy __
The Beer-lambert law states that the concentration of a substance is directly proportional to the amount of radiant energy absorbed
What is Bichromatic analysis ?
- monitors a reaction at two wavelengths
- used to correct for background interference
What is a bandpass ?
- range of wavelengths that reach the exit slit of a monochromator
- transmitted at a point equal to half the peak intensity transmitted
Define icterus
refers to the orange color imparted to a sample because of the presence of bilirubin
What is Kinetic Analysis ?
- change in the monitored parameter over time related to concentration
- measurements usually made very early in the rxn period
What is line spectrum ?
a discontinuous emission spectrum of elements in which emitted light bands cover very narrow (0.1 nm) range of wavelengths
What is Allen Correction ?
- multi-chromatic analysis of a rxn to correct for background absorbance
- two wavelengths, in addition to the MAX absorbance are monitored to subtract average background absorbance
The Beer-lambert law states that the __ of a substance is __ to the amount of radiant energy __
The Beer-lambert law states that the concentration of a substance is directly proportional to the amount of radiant energy absorbed
Define icterus
refers to the orange color imparted to a sample because of the presence of bilirubin
What is a bandpass ?
- range of wavelengths that reach the exit slit of a monochromator
- transmitted at a point equal to half the peak intensity transmitted
What is Bichromatic analysis ?
- monitors a reaction at two wavelengths
- used to correct for background color
What is line spectrum ?
a discontinuous emission spectrum of elements in which emitted light bands cover very narrow (0.1 nm) range of wavelengths
What is Kinetic Analysis ?
- change in the monitored parameter over time related to concentration
- measurements usually made very early in the rxn period
Define Molar Absorptivity
the absorbance of light, at a specific wavelength, divided by the product of the concentration of moles per litre and the sample path (cm)
Units for Molar Absorptivity
L/(mol x cm)
What is reflectance spectrophotometry ?
- a quantitative spectrophotometric technique in which light reflected off a surface of a colorimetric reaction is used to measure the amount of the reaction product
Define stray light
- Radiant energy reaching the detector that consists of wavelengths other than those defined by the filter/ exit slit of monochromator
- Causes false decrease in absorbance measurements/ deviation from Beer-lambert’s
How can stray light be detected ? How is it reduced ?
- detected by inserting a cutoff filter that does not transmit emission the light
If %T > 0 = stray light is present
- reduced by changing light source, sealing light leaks, realigning instrument components, cleaning optical surfaces, removing dust
Define turbidity (as an interferent)
- scatter of light in a liquid that contains suspended particles
ie. lipemia can make a sample turbid
What errors can occur with low %T ?
- greater imprecision
- substantial error when absorbance is calculated bc of compressed nature of the log scale
What errors can occur with low %T ?
- greater imprecision
- substantial error when absorbance is calculated bc of compressed nature of the log scale
What errors can occur with high %T ?
- detection system may have limited capacity to measure small differences between high levels of absorbance accurately
- can be overcome by diluting the sample and using a sample blank
How can spectral interferences be resolved ?
- using a sample blank
- lipemia can be cleared using ultracentrifugation or extraction
- turbidity can be reduced by dilution
List the 6 components of a spectrophotometer
- Polychromatic light source
- Entrance slit
- Monochromator
- Exit slit
- Cuvette
- Detector
List 2 methods used to correct for spectral interferences
- Bichromatic analysis
- Allen correction
How can spectral interferences be mitigated ?
- using a sample blank
- lipemia can be cleared using ultracentrifugation or extraction
- turbidity can be reduced by dilution
How can spectral interferences be mitigated ?
- using a sample blank
- lipemia can be cleared using ultracentrifugation or extraction
- turbidity can be reduced by dilution
What errors can occur with high %T ?
- detection system may have limited capacity to measure small differences between high levels of absorbance accurately
- can be overcome by diluting the sample and using a sample blank
List the 6 components of a spectrophotometer
- Polychromatic light source
- Entrance slit
- Monochromator
- Exit slit
- Cuvette
- Detector
What errors can occur with low %T ?
- greater imprecision
- substantial error when absorbance is calculated bc of compressed nature of the log scale
List 2 methods used to correct for spectral interferences
- Bichromatic analysis
- Allen correction
What 3 materials can Cuvettes be made of ?
- Borosilicate glass; 320 - 950nm
- Silica quartz; < 320nm
- Plastic; all wavelengths
What is the difference between photomultiplier tubes and photodiodes ?
Photomultiplier tubes:
- detect and amplify photons of light that hit its surface
- composed of photon sensitive cathode, series of DYNODES that amplify signal, and an anode
Photodiodes:
- semiconductors that change their charged voltage when hit by a photon
- generate a proportional current
- less sensitive; DOES NOT AMPLIFY signal
Describe how photodiode arrays work
- prism/ grating disperses light after the cuvette, and onto the array
- each photodiode is calibrated for a specific wavelength
- when a photon hits surface of a charged coupled device (CCD), electrons are released and quantitated
How can wavelength accuracy be confirmed ?
Using standard absorbing solutions OR filters with absorbance maxima of known wavelengths
How can linearity of detector be confirmed ?
- Using solid glass filters OR a solution known to follow Beer-lambert’s law ie. Copper sulphate
- Non-linear response can be due to faulty detector or stray light
How is a reflectance spectrophotometer calibrated ?
Using a white surface = reflects all incident (Io) light
Black surface = calibrates for zero reflectance
Dry slide chemistry is an example of __. Dry slides are composed of __ layers.
Dry slide chemistry is an example of reflectance spectrophotometry. Dry slides are composed of multiple layers.
Describe dry slide chemistry
- patient sample is applied on a spreading layer
- as the sample moves down through the layers, it can become altered
- the sample is impregnated with dry chemical reagents in the reagent layer - coloured product
- light is directed at the reagent/ indicator layer and the amount of diffuse light measured = [analyte]
“The detection and measurement of a decrease in the intensity of an incident beam of light as it passes through a solution of particles”
Turbidimetry
“The measurement of emitted light that occurs when a molecule absorbs light at one wavelength and reemits it light at a longer wavelength”
Fluorometry
What is an advantage of using fibre optics in spectrophotometers?
instruments can be miniaturized
Which light source can provide light in the visible spectrum?
Tungsten
Which light source can provide light in the UV spectrum?
Deuterium
Which of the following best describes photomultiplier tubes?
a.
PMTs are made of small silicon cells that when light enters release a proton.
b.
PMTs are made of small silicon cells that when light enters release an electron.
c.
PMTs are made of a cathode that can absorb radiant energy and release electrons in proportion to the radiant light.
d.
PMTs are semiconductor that change their charged voltage upon being struck by light; the change is converted to current and measured.
c.
PMTs are made of a cathode that can absorb radiant energy and release electrons in proportion to the radiant light.
cross out
Which of the following substances is suitable to use as a 100% reflectance standard?
a.
cobalt II sulfate
b.
barium sulfate
c.
silver I oxide
d.
silica dioxide
b.
barium sulfate
Which of the following actions may reduce stray light?
a.
reset 100% absorbance
b.
verifying linearity
c.
reset percent transmission (%T) to 100
d.
realign instrument components
d.
realign instrument components
How many wavelengths must be checked to certifying the accuracy of spectrophotometer with a grating?
2 wavelengths
Which of the following wavelength selectors may be found in a photometer?
a.
diffraction grating
cross out
b.
Wratten filter
cross out
c.
prism
cross out
d.
echelette
Which cuvette type is best for use below 320 nm?
a.
quartz
b.
polystyrene
c.
polyacrylate
d.
glass
a.
quartz
What is the function of an exit slit?
a.
to determine the band pass
b.
focus the light on the detector
c.
disperses light into its spectral form
d.
to make light parallel
a.
to determine the band pass
Which wavelengths of light (nm) will be transmitted from a interference filter designated for 550 nm?
a.
275, 550
b.
275, 550, 1100
c.
550, 1100
d.
550
b.
275, 550, 1100
Which of the following describes the function of a Wratten filter?
a.
selectively reflects the desired wavelength and absorbs all other wavelengths.
b.
reflects some radiant energy but allows other wavelengths to penetrate and reflect off a second surface.
c.
selectively transmits the desired wavelength and absorbs all other wavelengths.
d.
disperses radiant energy from the light source into a spectrum.
c.
selectively transmits the desired wavelength and absorbs all other wavelengths.
Which of the following substance is suitable for a 100% reflectance standard? (multiple answers)
a.
cobalt II sulfate
b.
barium sulfate
c.
silica dioxide
d.
silver I oxide
e. magnesium carbonate
b.
barium sulfate
AND
e. magnesium carbonate
Which is the best description of electronic transition?
a.
the movement of an electron from a lower energy state to a higher energy state
b.
the movement of an proton from one element to another
c.
the movement of an electron from one element to another
d.
the movement of an electron from a higher energy state to a lower energy state
a.
the movement of an electron from a lower energy state to a higher energy state
Which best describes the composition of a Wratten filter?
a.
one or more layers of colored glass
b.
colored gelatin sealed between two glass plates
c.
a glass plate coated with silver
d.
a glass plate coated with gold
b.
colored gelatin sealed between two glass plates
