Spectrophotometry Flashcards
Ultraviolet
<400nm
Visible light
400-700 nm
Infrared
<700 nm
Planck’s Formula
E=hv
relationship between wavelength and energy
inversely proportional to energy and frequency
shortest wavelength = __ energy frequency
↑ energy frequency
longest wavelength = __ energy frequency
↓ energy frequency
the concentration of a substance is directly proportional to the amount of light absorbed and is inversely proportional to the logarithm of transmitted light
Beer-Lambert’s Law
A=abc
what is ‘a’?
molar absorptivity
A=abc
what is ‘b’?
light path
A=abc
what is ‘c’?
concentration
Beer’s formula:
A= 2-log%T
(2 log Tayo after beer)
formula to determine the concentration of an unknown analyte
Cu= AuCs/As
(AUq CaSe AmpangetS)
distilled water, rgt, sample; sets the spectrophotometer to 0 absorbance
blank
- corrects absorbance caused by the color of REAGENTS
- used to 0 the instrument before measuring test samples and other blanks
reagent blank
used to subtract the intrinsic absorbance caused by hemolysis, icterus, turbidity, or drug interference
sample blank
substance of KNOWN purity and concentration; used to determine the concentration of the unknown analyte
a. standard
b. control
a. standard
- soln containing various analytes with known target values;
- analyzed with patient samples to monitor analytical performance (accuracy, precision)
- aka QC sample
a. standard
b. control
b. control
values provided by the manufacturer
a. assayed
b. unassayed
a. assayed
values determined by the laboratory
a. assayed
b. unassayed
b. unassayed
provides polychromatic light
light source
deuterium/hydrogen
a. UV, non-colorimetric
b. UV, visible light, colorimetric
c. visible, near IR
a. UV, non-colorimetric
Xenon/Mercury
a. UV, non-colorimetric
b. UV, visible light, colorimetric
c. visible, near IR
b. UV, visible light, colorimetric
tungsten, LASER
a. UV, non-colorimetric
b. UV, visible light, colorimetric
c. visible, near IR
c. visible, near IR
meaning of LASER
light amplification by Stimulated Emission of Radiation
prevents stray light
entrance slit
wavelength selector/isolator
monochromator
continuous, non-linear spectrum, better separation for high-frequency light
a. prism
b. diffraction gratings
a. prism
continuous, linear, uniform separation of wavelengths; most commonly used
a. prism
b. diffraction gratings
b. diffraction gratings
controls bandpass;
narrow fraction of the spectrum to reach the cuvette
exit slit
sample cell; may be round or square
cuvette
quartz/fused silica cuvette
a. UV-IR
b. UV-visible
c. visible; not for UV work
a. UV-IR
plastic cuvette
a. UV-IR
b. UV-visible
c. visible; not for UV work
b. UV-visible
glass cuvette
a. UV-IR
b. UV-visible
c. visible; not for UV work
c. visible; not for UV work
converts the transmitted light energy into an equivalent amount of electrical energy
photodetector
processes the electrical signal, performs mathematical operations, and displays the output
read-out device
Spectrophotometer QA:
if the WAVELENGTH is set to the actual one selected by the monochromator; checked using didymium glass or holmium oxide
wavelength accuracy
Spectrophotometer QA:
done using glass filters and solutions that have known ABSORBANCE values
absorbance check
Spectrophotometer QA:
A change in concentration results in a STRAIGHT LINE calibration curve (Beer’s law)
determined using neutral density filters and dichromate solution
Linearity
Spectrophotometer QA:
any wavelength outside the band of interest; causes absorbance error and loss of linearity; detected using sharp cut off filters
stray light
