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
This must be true in order for the Allen correction to be valid
background absorbance must be linear over the wavelength range
same essential components of a spec
radiant light source
wavelength selector
slits
cuvette
detector
readout display
light source for visible light
tungsten and tungsten halogen (quartz halogen)
- both continuous
- tungsten = 360 to 950
- quartz halogen lasts longer and higher intensity than tungsten alone
light source for UV
hydrogen deuterium
- cont
- 220 to 360
- deut more stable than hydrogen
mercury vapour
- discont
- 313, 365, 405, 336, 546
- low or high pressure; HPLC
visible & UV light sources
light emitting diodes
- cont and discont
- infrared, vis, UV, wh LED
lasers
- discont
- infrared, vis, UV
- expensive
- high intensity
- monochromatic light
these provide a narrower range of incident light wavelengths and are adjustable
monochromators
monochromators require two slits:
an entrance slit which focuses the light on the grating or prism and an
exit slit that determines the range of wavelengths, bandpass, of the exiting light
how is bandpass calculated?
Bandpass is calculated
by determining the difference between the maximum and minimum wavelengths at one half the height of
the peak transmission
prism
non-linear dispersion of light into a continuous spectrum by refraction
grating
Linear dispersion of light into multiple mini spectrums
A large number of parallel spaced grooves on a surface
Borosilicate glass cuvettes
used for measurements between 320 to 950 nm
Silica quartz cuvettes
used for measurements of light less than 320 nm
these cuvettes are available for all wavelengths
plastic
these tubes detect and amplify the photons of light that hit its surface
photomultiplier tubes
semi-conductors composed of light-sensitive materials that change their charged voltage upon photon impact and generate a proportional current
photodiodes
- less sensitive than photomultiplier tubes bc they do not amplify the signal
how is wavelength accuracy checked?
by using standard absorbing solutions or filters with absorbance maxima of known wavelengths
- can also be performed by
substituting a continuous light source with a discontinuous source with a strong emission line
T or F. At least 2 wavelengths should be verified in instruments with prisms and at least 3 wavelengths in instruments with grating
F! 3 for prisms; 2 for gratings
The linearity of the detector response can be checked using…
solid glass filters or by using a solution
known to follow the Beer-Lambert law, such as copper sulphate
a nonlinear response may be the result of a …
faulty detector or stray light
what is stray light?
radiant energy that reaches the detector at wavelengths other than those indicated by the
exit slit of the monochromator
this decreases absorbance measurements and causes deviation from the Beer-Lambert law and it can be detected by inserting a cutoff filter that does not transmit the
emission light
stray light
- if the %T is greater than 0%, stray light is present
how can stray light be reduced?
changing the light source
sealing light leaks
realigning instrument components
cleaning optical surfaces
maintaining a dust-free environment
this measures the amount of light reflected off a flat test surface
reflectance spectrophotometry
how does a reflectance spec work?
- mono light directed towards a test surface (via diffraction grating or light emitting diodes)
- light hits test surface
- colour intensity of the test surface increases with the concentration of the analyte, absorption increases and diffuse reflectance decreases
- single wavelength of the diffuse reflected light is selected and directed onto a photodetector which relays the result to a readout device
how is a reflectance spec calibrated?
using a white surface, similar to the test surface, that reflects all incident light
- standards composed of magnesium carbonate or barium sulphate may be used for this purpose
- black standard may be used to calibrate zero reflectance
how can stray light be corrected in reflectance spec?
by subtracting light detected during a zero calibration from the test value
DR = log (R0/Rtest)
The reflection density (DR) is a
measure of the amount of light absorbed by the chromophores on the test surface. The DR is the logarithm of the ratio of the light reflected by the white standard (R0) to the light reflected by the surface (Rtest)
T or F. The relationship between analyte concentration and reflectance is not linear
T
- microprocessor is
required to transform the data into a linear format
- both the Kubelka-Munk equation and the Clapper Williams transformation are commonly used to convert the data
where is reflectance spec used?
n semi and fully automated urinalysis instruments such as the Clinitek Status and the IRIS
- this technique is used in dry slide instruments such as the Ortho Diagnostic Vitros and FujiFilm DryCHEM NX500
