1. Describe the mechanism by which atoms and molecules absorb light. What
atomic/molecular characteristic determines the wavelength of light that is absorbed?
2. Calculate absorbance from %T. Use Beer's law to relate the absorbance or %T of a
solution to the concentration of the absorbing compound. List the assumptions inherent in
3. List the components of a spectrophotometer. List examples of each component. Describe
how each component works.
lamp (visible and UV)
monochromator (fixed wavelength and variable wavelength)
detector (PMT and photodiode)
4. Describe bichromatic analysis including the principle, arrangement of components and
advantages. Extrapolate the principle to multiple-wavelength spectrophotometry. List
examples of common interferences that can be corrected using bichromatic analysis.
5. Describe check systems for verifying the performance of spectrophotometer components.
List the components that require checking, and describe the errors that would be caused by
malfunctions. Describe the reference components used for checking.
6. Define band-width (band-pass) and stray light, and describe their effects on absorbance
7. Define fluorescence. Describe the atomic mechanisms that result in fluorescence.
Describe the relationship between amount of fluorescence emitted and concentration of the
8. Define nephelometry. Describe the relationship between amount of light scattered and the
concentration of particles. List the limitations of using nephelometry.
9. Compare the components of a fluorometer and a nephelometer to the components of a
spectrophotometer. Contrast their arrangement and functions.
10. Define reflectance spectrophotometry. Correlate it to transmission spectrophotometry.
I. Wavelength absorbed by an atom or molecule corresponds to energy levels available to
the valence electrons.
K shell (max 2 e-)
L shell (max 8 e-)
M shell (max 18 e-)
1st comfort level @ 8 e-
The energy and wavelength of a photon are inversely related to each other.
Colors perceived by the eye correspond to certain ranges of wavelengths.
II.Beer's law relates concentration of solute to the transmission of light through its solution.
(opaque cuvet or shutter)
Defined as 0% T
(cuvet with solvent)
= I/Io X 100
Each successive "layer" of solute absorbs a constant % of the light that is incident on it
Thus, %T is a logarithmic function of solute concentration
1. Define a new term: Absorbance = A = Abs
A = - log I = -[log I - log Io] = log Io - log I
when Io is set at 100 % then log Io = 2 and I is %T
∴ A = 2 - log I = 2 - log %T
2. Beer's Law: A = abc
where A = absorbance
c = concentration
b = length of light path thru solution (i.e., width of cuvet)
a = proportionality constant
also called extinction coefficient or molar absorptivity
In short: A ∝ concentration
Assumptions: only 1 chromogen and only 1 wavelength of light
A. Light source = lamp
Must emit the desired wavelength with enough intensity to reach the detector
Types: tungsten (& tungsten halogen) → ~320 to >1000 nm = visible & infrared
xenon → ~200 to ~800 nm = UV and visible require HV
deuterium & hydrogen → ~200 to ~450 nm = UV power supply
mercury → several sharp spikes in UV range
Chromogen ABSORBS λ that corresponds to energy level of its e-,
and TRANSMITS the rest of the wavelengths
Beer's law : concentration ∝ absorbance
∴ select λ that is absorbed in order to measure concentration
λ absorbed is complementary to the λ that is transmitted
yellow and blue are complementary to each other I O
red (and violet) are complementary to green
1. Fixed wavelength types of monochromators: G
Colored glass or plastic
absorbs λ complementary to its color (appearance)
transmits λ that is even multiple of distance between its sides
If distance between the surfaces
is an even multiple of λ, then
reflecting waves stay in phase →
constructive interference →
If distance between the surfaces
is NOT an even multiple of λ,
then reflecting waves get out of
phase → destructive
Note: several λ's will be even multiples; called 1st, 2nd, 3rd order wavelengths;
remove them with cut-off filters before the monochromatic light hits the
2. Variable wavelength types of monochromators spread the λ's of incident light
into a continuous spectrum of different colors;
then the desired color (λ) is selected by passing it to the cuvet through an exit slit.
obsolete because short λ's are spread out nicely (can pick out the λ
you need), but long λ's are crammed together (can't select just one
b. diffraction grating "bends" light around sharp edged grooves inscribed
in a reflective surface
Short wavelengths are diffracted through only a small angle; long
wavelengths through a large angle;
λ's that overlap either reinforce each other (constructive
interference) or cancel each other (destructive interference);
∴ the incident light is spread out into one continuous spectrum
(like one rainbow from diffraction off of many water droplets)
C. Cuvet - holds sample in the light path; ∴ defines "b" in Beer’s Law A = abc
must transmit essentially all of the incident light
ordinary glass transmits > ~320 nm
special acrylic plastic transmits > ~320 nm
Fused silica or quartz glass transmits > 200 nm
must use "matched set" of cuvets, i.e., all have identical, minimal absorbances
(diameters, reflections, refractions, imperfections, etc.) and are clean (no left-over
reagents or soap, no fingerprints from handling)
D. Detector - transduces light energy into electrical energy
Light hits surface of selenium or cadmium oxide → dislodges e-'s → current or ∆
Photocell fatigue = all the available e- have been removed, ∴ surface is "blind"
Two common types:
photomultiplier tube (PMT) photodiode
multiplies and measures current measures drop in voltage
requires high voltage power supply
has dark current
IV. Bichromatic analyzer splits the light beam in two and selects a different λ for each
One λ is the absorption peak of the analyte (desired chromogen),
∴ Abs at this λ measures the concentration of the analyte
PLUS cross-over absorbance due to other interfering chromogens in the
Second λ is NOT absorbed by the analyte but IS absorbed by the interfering
chromogen, ∴ this absorbance indicates how much interfering chromogen is
The ratio of absorbances at the two wavelengths is must be known for the
Then subtract the interfering absorbance to determine the concentration of the
B. The function of the 2nd λ is to act as the "serum blank"
"Blank" = solution in a cuvet that does not contain specific absorbing substance,
e.g., you did not perform the color reaction.
ex: if reagent itself is colored, then total Abs =
Abs due to color reaction with the analyte plus Abs due to reagent
∴ set the cuvet containing reagent (in the same proportion) at 100 %T
= “reagent blank”
ex: if sample itself is colored, then total Abs =
Abs due to color reaction with the analyte plus Abs due to background
∴ set cuvet containing sample (in the same proportion) but NO COLOR-
REACTION at 100 %T = “serum blank”
Examples: hemolysis (red hemoglobin), jaundice (yellow
bilirubin), lipemia (turbidity due to triglycerides)
Example of calculations and assumptions:
analytical cuvet contains: serum blank cuvet contains:
Abs due to cuvet glass Abs due to cuvet glass
Abs due to colored reagent Abs due to colored reagent
Abs due to sample color Abs due to sample color
Abs due to reaction-chromogen
Total Abs in analytical cuvet Total Abs in blank cuvet
Set blank cuvet = 100 %T = 0 Abs, ∴ all Abs in blank cuvet has been
subtracted leaving only Abs due to the desired analytical reaction
V. Performance checks
Principle: each component can malfunction and cause errors in the absorbance reading.
A. Lamp check: Does the lamp emit sufficient light energy?
Select a wavelength where the lamp only emits a little bit;
can you set the meter reading at 100 %T?
B. Monochromator check: Is the λ transmitted to the cuvet the same as the λ reading
on the knob?
Place a substance with known absorbance peaks and valleys in the cuvet;
do the known maximum and minimum peaks and valleys correspond with the
reading on the knob?
primary wavelength calibrators: didymium and holmium oxide filters
secondary calibrators: any solution with known peaks and valleys in its
ex: NiSO4 or KMNO4 or CoCl2
1. Used in variable λ spectrophotometers to select the desired λ from the
∴ wide slit allows many surrounding λ's to reach the cuvet = wide
narrow slit allows only 1 (or few) λ's to reach the cuvet = narrow
2. Is the absorbance reading of a known calibrator as high as expected?
Wide bandpass "averages" (integrates) the reading at the desired λ
with all the readings at surrounding λ's → low overall reading
Exit slit is wider than expected (causing shallow peaks and valleys) if
lamp is too dim
slit gears malfunction
slit becomes effectively wide by having dirt/dust collect on its edges
C. Cuvet Check: Is identical absorbance reading obtained when one solution is read in
Sources of error include: warped glass
cuvet diameter is not the same
dirt inside (absorbs or reflects light)
dirt outside (absorbs or reflects light) - including
D. Detector Check: Is the absorbance reading of a known calibrator equal to the
Primary calibrators are "neutral density" filters
graded amounts of gray dye with known absorbance values
Sources of error
dirt or chemical film on the surface of the detector (absorbs or reflects
wrong or variable voltage to the dynodes
"photocell fatigue" = too bright light removed all the surface e-; wait a few
I. Atomic mechanism of fluorescence
A fluorescent molecule absorbs a specific λ of light which raises an e- to a higher orbit
(same as spectrophotometry)
10-8 to 10--4 seconds later, the e- falls back to its ground state orbit and the energy is
released as emitted light.
During the 10-8 to 10-4 seconds, the electron vibrates and rotates in its chemical bond
which uses up some energy.
∴ the λ of emitted light has less energy (longer λ) than the exciting light (shorter λ)
λemission > λ excitation
The exciting light is collimated into a narrow beam (same as spectrophotometer), but the
emitted light leaves the cuvet in all directions
∴ the emission monochromator & detector are at right angle to the exciting light beam
Need both primary (excitation) and secondary (emission) monochromators
III. Calculating concentration:
Every excited molecule emits fluorescent light,∴ fluorescence ∝ concentration
Fluorescence is ~ 1000 x more
sensitive than spectrophotometry
but exciting light must penetrate
the whole cuvet to have
Can detect the emitted light from
a tiny amount of fluorescent
molecule because 0 concentration
→ no fluorescence = blackness
I. Nephelometer (or turbidimeter) is used to measure turbid suspensions.
When cuvet contains insoluble particles, incident light is scattered in all directions.
Then less light reaches the detector of a spectrophotometer → pseudo-absorbance
∴ amount of turbidity appears to follow Beer's law; this is called Turbidimetry
Used to measure insoluble product, e.g., aggregates of Ag-Ab complexes (precipitin
In a nephelometer, the scattered light is measured at an angle (like fluorometry) except
photon was only scattered, not absorbed, ∴no change in λ ∴ no secondary
Amount of light scattered ∝ concentration of insoluble particles;
this is called nephelometry.
III. Calculating concentration
In both turbidimetry and nephelometry, there is a very limited range of concentrations
where light scatter is a function of particle concentration. The principle limiting factor is
how far the incident light can penetrate the turbid suspension.
Don't forget that suspensions tend to settle out of solution, ∴ must have a well-mixed
I. Atomic Principle:
When colored analyte sits on the surface of an opaque support, then it absorbs the same λ
as if it was in solution and reflects the complementary λ's.
Concentration ∝ inverse log of
amount of light reflected
∴ follows an equation that resembles
(This also works if the surface analyte is fluorescent, then it emits light the same as if it
was in solution , and concentration ∝ amount of fluorescent light.)
Reflectance photometry is commonly used in instruments that measure antigens because
the antigen-antibody complex formed in the analytical reaction can be adsorbed onto a
filter while the unreacted materials flow through the filter to waste.
Also commonly used in urine dipsticks where numerous solutes in a urine sample are
tested in individual color reactions on separate pads of reagents tests