Bios 532
Laboratory Module on UV-Visible
Light Absorption Spectroscopy
Absorption Spectroscopy
The Beer-Lambert Law
A=lc
Where A is absorbance or optical density
(no units, since A = log [I0/I]);
 is the molar absorptivity with units of (L mol-1 cm-1);
l is the path length of the sample - that is,
the interior dimension (cm) of the cuvette
in which the sample is contained, parallel to the light path;
c is the concentration of the compound in solution,
expressed in (mol L-1).
Molar Absorptivity
A = lc
 is a measure of the amount of light absorbed
per unit concentration at a particular .
Molar absorptivity is a constant for a particular substance,
so if the concentration of the solution is halved, so is the
absorbance at sufficiently dilute concentrations.
A
concentration
A = bc
Limitations
this relationship fails at extremely high concentrations
• electrostatic interactions between molecules in close proximity
• changes in refractive index at high analyte concentration
• shifts in chemical equilibria as a function of concentration
A
concentration
Limitations
Absorbance (A) versus the measured transmission (%T):
A sample which allows 100% of the selected light to be transmitted
clearly has zero absorbance.
A sample which allows 0% transmittance exhibits
infinite absorbing power: ∞ = A .
These limits are properly accounted for through the following
logarithmic relationship between absorbance and percent
transmittance:
A = - log (%T /100)
Limitations
A = - log (%T /100)
Errors in %T readings which are too low (e.g., below ~12%) or too
high (e.g., above ~70%) are significantly magnified in their
corresponding A values.
 Design experiments so that only samples with %T values above
12% and below 70% are prepared and measured.
This corresponds to solutions with absorbance values A between ~
0.16 and 0.92.
Review of these principles can be found on-line in the US Naval Academy Dept. of Chemistry
General Chemistry Laboratory Manual:
http://www.chemistry.usna.edu/manual/ApdxI.pdf.
Spectroscopic light sources - terms
• monochromatic light - light of a single, constant wavelength.
• coherent light - wavelengths are in phase in space and time.
• white light is a mixture of all the colors of the visible spectrum,
which includes light with wavelengths of 400 - 700 nm.
Ordinary light (emitted from the everyday light bulb, the sun, a
candle) will consist of wavetrains of unrelated phases,
frequency and polarization in all directions.
In contrast, laser light is monochromatic, directional and
coherent.
Light Source in the Shimadzu Specs
1. The halogen visible light source - typical spectral emission range
for a halogen lamp is ~ 300 -1100 nm.
2. The deuterium UV light source - typical spectral emission range
is ~ 180 - 500 nm.
3. Switching between the two light sources is performed
automatically by the spec, according to a pre-selected switching
wavelength that has been chosen at installation. The switching
wavelength is usually somewhere between 280 - 390 nm.
Cuvettes
Ordinary glass is partially transparent to UV but is opaque to
shorter wavelengths while Silica or quartz glass, depending on
quality, can be transparent even to vacuum UV wavelengths.
Ordinary window glass passes about 90% of the light above 350
nm, but blocks over 90% of the light below 300 nm.
1. Use quartz cuvettes for UV experiments.
2. Use quartz, glass, or plastic for visible wavelengths.
Difference Spectroscopy
• Two solutions are compared - often start out identical.
• All common spectral features cancel out.
• Reference solution is unperturbed.
• The sample solution is varied by additives.
• Double-beam operation - the spec does the subtraction (the
Shimadzu specs have a beam chopper).
Difference Spectroscopy
The chopper resembles a fan that allows light to pass for a
characteristic period of time, then blocks the light for the same time
period. The unblocked signal is reference + sample, the blocked signal
is reference only. The difference between these two signals (blocked
and unblocked) must be the desired analytical signal.
•
beam chopper - an optical chopper is a mechanical or
electromagnetic device for passing and then interrupting a beam
of light for a known brief interval. Examples include tuning
forks, rotating shutters and the more sophisticated Kerr cells.
•
monochromator - an instrument for isolating narrow portions of
the spectrum The spectrum of any light source is formed by a
prism or grating, and an exit slit placed in the spectrum selects a
narrow band of wavelengths for emission. By moving the
spectrum of a source internally past the slit, the color of the
emitted light can be varied at will. As most monochromators
emit several percentages of unwanted light -- either white or of
the wrong wavelength -- along with the desired wavelength, two
monochromators often are used in tandem, both being set to
transmit the same wavelength. In this way the percentage of
unwanted light can be reduced drastically.
monochromators
1. Grating monochromator - uses a diffraction grating as the
dispersive element that is a ray of fine, parallel, equally spaced
reflecting or transmitting lines that mutually enhance the effects
of diffraction to concentrate the diffracted light in specific
directions determined by the spacing of the lines and by the
wavelength of the light.
2. Crystal monochromator - the crystal lattice serves as a 3dimensional diffraction grating that separates light by
wavelength (think prism).
Factors other than sample path length and
sample concentration that can affect absorbance
 reflections at the cuvette interface
 scratches, fingerprints on the cuvette
 absorptive materials in buffer (non-sample)
 fluorescence (increases apparent intensity)
 high sample concentrations
 change in the chemical properties of the sample
Hemoglobin
A
B
1
2
Fe
D
C
1
2
Fe Fe2+ (Fe(II), ferrous)  Fe3+ (Fe(III), ferric)
minimum oxidation number: -2
min. common oxidation no.: 0
maximum oxidation number: 6
max. common oxidation no.: 3
Iron metal reacts in moist air by oxidation to give a hydrated iron
oxide. This does not protect the iron surface to further reaction
since it flakes off, exposing more iron metal to oxidation. This
process is called rusting and is familiar to any car owner. Finely
divided iron powder is pyrophoric, making it a fire risk.
Methemoglobin
Fe(II)-heme is oxidized to Fe(III)-heme to form metHb.
MetHb does not bind O2.
At low pH, H2O occupies the space between the Fe and the
distal histidine - aquomethemoglobin.
At high pH, Fe binds OH- - hydroxymethemoglobin.
oxy-Hb
met-Hb
When a ligand
is bound to Hb,
the heme iron
is 6-coordinated.
Visible Spectrum of Methemoglobin
ß
oxyHb
low spin

500
metHb/CN
low spin
aquometHb
high spin
deoxyHb
high spin
630
490-510
ß

600-630

UV


High Spin State vs. Low Spin State
Elemental Fe
Fe2+ (ferrous)
Fe3+ (ferric)
metHbIf there
were no
ligands
[Ar]3d64s2
[Ar]3d6
[Ar]3d5
low pH+H2O
high pH+OH -
1/2
1/2
1/2
1/2
1/2
5/2 high spin
1/2
1/2 - 1/2
1/2 - 1/2
1/2 low spin
Absolute Spectrum of Methemoglobin

ß
500
high
spin
low
spin
630
490-510
ß

600-630
Inositol hexaphosphoric acid (IP6 or phytic
acid) binding to met Hb:
Physiological relevance - the binding of
organic phosphates to ferrohemoglobin
results in a decrease in oxygen affinity,
linking erythrocyte metabolism to gas
transport.
IP6 binding causes a change in spin character of
ß chain hemes.
p-hydroxymercuribenzoate
(pMB) binding to met Hb
ß - Cys 93
92
145
93
146
94
 subunit in red
pMB binding changes the spin character of
met Hb
Remember
 KCN, NaN3, PMB, NaF are all toxic. (NaF eats glass when wet.)
 Use a reference cell or a baseline reading for subtracting buffer.
 Save all files in the directory UVPC:Data:Bios532.
 All buffers, chemicals are pre-prepared, use only what you need.
 Store buffers, chemicals, protein solutions in 4 C between labs.
 Clean cuvettes with ethanol, air dry.
 Rinse syringes with millipore H2O.