CHEM Light Measuring Systems Flashcards
Formula for Absorbance
2 - log%T
NOTE: The relationship between absorbance and %T is LOGARITHMIC
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
How do spectrophotometers measure transmittance ?
By comparing the signal generated by the transmitted light (Is) to the signal of the incident light (Io)
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 requirements/ limitations of the Beer-Lambert law for measuring Absorbance
- monochromatic light (Io)
- solvent has insignificant absorbance to analyte
- 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 __.
In reflectance spectrometry, the relationship between analyte concentration and reflectance is LOGARITHMIC
What can be used to correct for spectral interferences during analysis ? How ?
-
Bichromatic Analysis:
- 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) -
Allen Correction:
- two wavelengths PLUS 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
Define icterus
orange color in a sample because 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
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 of desirable light
- If %T > 0 = stray light is present
- reduced by changing light source, sealing light leaks, realigning instrument components, cleaning optical surfaces, removing dust
Examples of turbidity (as an interferent).
- lipemia
- high cell counts
NOTE: causes scatter of light in a liquid that contains suspended particles
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 ? How can it be resolved ?
- detection system may have limited capacity to measure small differences between low levels of absorbance accurately
- can be resolved by diluting the sample and using a sample blank
How can spectral interferences be resolved ? (before analysis)
- using a sample blank
- lipemia = ultracentrifugation or extraction
- turbidity = dilution
List the 6 components of a spectrophotometer
- Polychromatic light source
- Entrance slit
- Monochromator
- Exit slit
- Cuvette
- Detector
How is a reflectance spectrophotometer calibrated ?
- White surface = reflects all incident (Io) light
- Black surface = calibrates for zero reflectance
“The detection and measurement of a decrease in the intensity of an incident beam of light as it passes through a solution of particles” is called what ?
Turbidimetry
“The measurement of emitted light that occurs when a molecule absorbs light at one wavelength and reemits light at a longer wavelength” is called what ?
Fluorometry
What is an advantage of using fibre optics in spectrophotometers?
instruments can be miniaturized
What substance are suitable for a 100% reflectance standard? (choose all that apply)
clear substances
ie. barium sulfate and magnesium carbonate
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
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 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
Define Fluorescence
Absorption (excitation) of light at wavelength and and re-emission (emission) of light at a longer wavelength by a molecule
Explain how fluorescence works
- Energy from high energy wavelengths is absorbed: electrons transition from ground state (0) to an excited state (1)
- Radiation-less Vibrational Deactivation (RVD) causes a loss in some energy
- When electrons drop from the excited state back to ground state, fluorescence is emitted at a lower frequency
What is RVD in fluorometry ?
Radiation-less Vibrational Deactivation - some energy loss before electrons return to ground state in fluorescence
Describe Stokes Shift in fluorometry
The difference between the maximum excitation wavelength and maximum emission wavelength
How is background signal from light scatter minimized in fluorometry ?
cuvette and detector are at right angles
In fluorometry, what is the Inner Filter Effect ? How can it be minimized ?
- It occurs when the sample absorbs >2% of the exciting light; non-linear fluorescence response that does NOT follow Beer’s Law
- Minimize by placing the excitation monochromator slit closer to a corner of the cuvette; reduces the amount of light absorbed by detector
