360 - Light Measuring Systems Flashcards
velocity of light in a vacuum
3x10 ^10 cm/s
polychromatic light
composed of multiple wavelengths and is commonly called white light
when poly light hits coloured solution = some wavelengths absorbed and complementary wavelengths transmitted
the amount of light transmitted through a solution can be quantified using the ______ property of light
particle
when photon hits light-sensitive material, e- moves to higher energy state = releases a free e-
Transmittance =
[Is/Io] x100%
the relationship between absorbance and transmittance is ..
logarithmic
A =
2-log%T
T or F. Light varies directly with frequency and inversely with wavelength
T
The amount of light transmitted through a solution can be quantitated using which property of light?
particle
Which conditions must be met in order for Beer-Lambert to be applicable?
- monochromatic incident light
- solvent absorption insignificant to analyte of interest
- analyte must follow B-L law
- standard and unknown must be measured in same cell
- concentration range that obeys B-L must be established
- absorbance of unknown must be less than standard; dilution!
- optical interferent not present
- chemical rxn must not occur between analyte of interest and another molecule
- parallel cuvette sides
- no stray light
At low %T, there is _______ imprecision when transmitted light is measured
greater
- substantial error when corresponding absorbance is calculated because of the compressed nature of the log scale
T or F. . At high %T, the error of calculating absorbance is relatively large to the absolute value of absorbance
T!
What errors occurs when there is high absorbance
detection system may have a limited capacity to measure small differences between high levels of absorbance accurately
- The high absorbance may be overcome by diluting the sample as well as using a sample blank
Hemoglobin has a significant absorbance at this range
500 to 600 nm in the visible spectrum
T or F. The presence of hemoglobin and bilirubin in a sample tend to cause negative interference
F! positive
the amount of light is absorbed by the sample increases
What happens if high levels of lipemia or VLDL is in a sample?
light is scattered and less transmitted light reaches detector = giving APPEARANCE of increased absorbance
When performing an endpoint reaction, where the reaction is monitored at a single time point, a ________ _________ can be used to subtract endogenous spectral interference
sample blank
kinetic rxn
measurements made overtime
- initial reading is taken when there is almost no colour development; thus, any colour at this time point is primarily caused by endogenous spectral interference
- second reading is
taken a short time later when only a small amount of colour has formed; response of absorbance
vs time is still linear
- the subtraction of the first reading from the second reading = delta
absorbance. Thus the kinetic reaction can be self-blanking
- lipemia can be cleared by ultracentrifugation
or by extraction, i.e. Lipoclear tubes
this can be used to correct for spectral interference when the maximum absorbance of the analyte or chromogen is the same as the spectral interferent
bichromatic analysis
- reading is taken at max absorbance wavelength (A1)
- second measurement near wavelength where analyte minimally absorbs light (A2)
- standard curve is based in either A1-A2 or ratio of the two measurements (A1/A2)
What is the Allen correction?
uses absorbance readings at three wavelengths = primary max absorbance and two additional wavelengths (equal distance from the Amax)
- corrected absorbance = sum of the two peripheral absorbance measurements is divided by two and subtracted from the max absorbance
