Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 173 (2017) 965968

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Spectrochimica Acta Part A: Molecular and Biomolecular

Spectroscopy
journal homepage: www.elsevier.com/locate/saa

Editorial

UVVIS absorption spectroscopy: Lambert-Beer reloaded

Werner Mntele , Erhan Deniz
Institut fr Biophysik, Johann Wolfgang Goethe-Universitt Frankfurt am Main, Max-von Laue-Strae 1, D-60438 Frankfurt am Main, Germany

a r t i c l e i n f o a b s t r a c t

Available online 21 September 2016
UV-VIS absorption spectroscopy is used in almost every spectroscopy laboratory for routine analysis or research.
All spectroscopists rely on the Lambert-Beer Law but many of them are less aware of its limitations. This tutorial
discusses typical problems in routine spectroscopy that come along with technical limitations or careless
selection of experimental parameters. Simple rules are provided to avoid these problems.
2016 Elsevier B.V. All rights reserved.

A tutorial on UVVIS spectroscopy? At rst sight, this does not seem
to be necessary. We all use in our labs UVVIS spectrometers every day,
either as routine instruments or for research, and thus should know the
rules. Yet in more than 10% of the manuscripts in my inbox I see crude
violations of Lambert-Beer's law, either by wrong design of the experi-
ment or because the prerequisites to apply the basic laws to measure
transmission or absorption are not followed. Routine procedures bear
the risk that potential pitfalls are not considered.
Where are the problems? It starts with the fact that the terms
absorption and extinction are very often used synonymously and
used as labels at the ordinate of the spectrum plots. I wish we would be
more precise here. Absorption is dened as the process whereby the
light intensity from the measuring beam is diminished because molecules
in the sample undergo a transition from the ground state (usually the
singlet state S0 for molecules at room temperature) to an excited state
S1, S2, or higher. Extinction refers to the entire loss of light energy
upon passing through the sample. It includes absorption, but also light
scattering and reection, processes that have nothing to do with the
absorption process. The extinction is thus equal to or higher than the
absorption.
The numerical value obtained in a UVVIS spectroscopy experiment
by application of Lambert-Beer's law (historically more correct: the
Bouguer-Lambert-Beer Law):
I0
A logT log cd
I
T: transmission;
I0, I: intensity of the measuring beam before/after passing through
the sample;
: molar absorption coefcient;
c: concentration;
d: path length of the measuring beam in the sample.
Corresponding author.
E-mail address: maentele@biophysik.uni-frankfurt.de (W. Mntele).
is typically called absorbance , A, and plotted vs. the wavelength.
Spectroscopy purists plot the molar absorption coefcient vs. the
wavelength, and real hard core spectroscopists plot it vs. the wave-
number in cm 1.
Please note that both transmission and absorption are dened on the
basis of the ratio of two intensities, before and after the sample. No
matter what unit is used for I and I0, absorption and transmission are
dimensionless. For transmission, the value obtained is typically
multiplied by 100 and denoted as %T. For absorption, it is rather a bad
habit if spectroscopists use absorbance units, AU, or OD for optical
density units.
While these are merely semantic problems or badly dened physical
magnitudes, the pitfalls on the technical side are more relevant. When
we learnt about the use of the Lambert-Beer law in spectroscopy, we
also learnt that it is only strictly valid if some fundamental conditions
are fullled. The most relevant are:
strictly monochromatic measuring light;
homogeneous distribution of the molecules in the sample;
passage of the complete measuring beam through the sample;
absence of light scattering and of photochemical reactions in the
sample;
no re-emission of the absorbed light by uorescence;
an ideal detection and processing of the intensity values I0 and I.
Real life is not ideal, and so is not absorption spectroscopy. The rst
condition strictly monochromatic light is already hard to guarantee.
Our routine spectrometers typically use a tungsten-iodine light source
for the near-UV to near IR spectral range (e.g. 3801000 nm) and a
deuterium discharge lamp for the UV from about 200 to 380 nm. A ip
mirror is used to switch between the lamps. For a perfectly adjusted
optics, the absorbance values of a sample before and after ipping this
mirror should be identical, but most spectra (including the ones I see
in incoming manuscripts) show an offset. In order to obtain monochro-
matic light (a euphemism, because strictly monochromatic would

http://dx.doi.org/10.1016/j.saa.2016.09.037
1386-1425/ 2016 Elsevier B.V. All rights reserved.
966
mean that the intensity is zero), most instruments use a grating
monochromator that is moved by a stepper motor. The spectral
bandwidth of the output of this monochromator is a compromise be-
tween intensity and spectral bandwidth set by the mechanical width
of the monochromator output slit. Since 12 nm spectral bandwidth
can be easily obtained with still sufcient light intensity, this is not real-
ly a problem. The pitfalls come from the fact that any grating monochro-
mator uses the grating at a certain diffraction order: rst, second, third
and so on. The interference conditions of a grating state that at any out-
put wavelength the grating is set, there is also intensity at higher or-
ders, i.e. 2, 3 and so forth, albeit at much lower intensity. This
means that if your monochromator is set at blue-violet light at
400 nm, the measuring beam also contains light at 800 nm. Just imagine
that if your sample absorbs strongly at 400 nm but is transparent at
800 nm; the detector will not be able to correctly measure the residual
intensity at 400 nm because of the fraction of light at 800 nm detected.
Manufacturers of spectrometers cope with this physics of diffraction by
placing so-called order lters in the beam after the monochromator
that let the desired light pass and block or at least reduce the higher
order light intensity. Nevertheless, there are traces of unwanted light
in the measuring beam that limit the application of the Lambert-Beer
law. This phenomenon is frequently called spectral false light, in con-
trast to spatial false light that we will deal with below.
Another source of spectral false light is stray light in the monochroma-
tor. This means that a small but noticeable fraction of the incoming white
light will also appear at the output. For a well-designed monochromator,
stray light is on the order of 103 to 105 of the incoming light intensity.
Stray light is equally sensed by the photodetector and also limits the min-
imum measurable intensity or the maximum of measurable
absorbance.
A third source of spectral false light may occur if the sample is strongly
uorescent. Let us assume that the sample has a peak absorbance of 2
which means that only 1% of the measuring light is passing at this wave-
length. If the uorescence quantum efciency of the sample is high, a high
fraction of the absorbed photons (99% in our example) is re-emitted as
uorescence. Again, the photodetector does not care whether it sees the
residual intensity I (1% in our example) of the measuring light or the in-
tensity caused by the re-emitted photons at a wavelength shifted to the
red. If the detector is more sensitive for the uorescence wavelength,
the uorescence intensity can appear even higher than the transmitted
intensity. The precise estimation of this error source requires knowledge
about the geometry of the sample, the optics and the detector.
Molecules may diffuse and rotate while the molecule is in the excited
state; uorescence emission is thus not in the direction of the measuring
beam, but rather isotropic.
There are means to get rid of the spectral false light caused by
sample uorescence. The best solution is a second monochromator
between sample and detector, set at equal spectral resolution and
moved synchronously with the rst monochromator. This guaran-
tees that only the measuring light passes from the sample to the
detector. This luxury solution solves the problem caused by uores-
cent samples and the problem caused by stray light, but not the
higher order problem, unless order lters are included, too. The
cheaper solution is to increase the distance between the sample
and the detector and to use a parallel beam of light for the absorption
measurement. If so, the transmitted measuring light I is recorded
independent from distance r, while the intensity of the uorescence
light will decrease approximately with 1/r2 because it is emitted
almost isotropically. An even cheaper solution is dilution of the
sample. As you dilute, the transmitted intensity I will increase
while the emitted uorescence intensity will decrease.
Spatial false light has many origins. There are trivial ones, such as
incompletely lled cuvettes where the measuring beam partly
passes above the sample, and this fraction reaches the detector unat-
tenuated. There are badly adjusted cuvette holders that let part of
the light pass sideways. You may think that I report problems that
Editorial
are typical for rst-year students, but I have seen this for experi-
enced senior scientists in research labs. The so-called microcuvettes
that are popular because they require only small sample volumes are
prone to this problem, since they have only a narrow slit for the sam-
ple with glass or plastic walls left and right. The slightest shift of
these cuvettes causes the measuring beam to pass partly through
the glass walls. The solution can be as trivial as the problem: tale a
black felt pen and paint the glass walls left and right from the sample
part until they are intransparent. There are also blackened cuvettes
available; while they are more expensive, their use should be much
preferred in particular for very small sample volumes.
Light scattering in the sample is also a false light problem. The ideal
sample in the sense of the Lambert-Beer law is a homogeneous solution
without light scattering. Many chemical, biochemical or biological
samples are suspensions or textured structures that exhibit light
scattering. It is not always necessary to describe light scattering
quantitatively, but spectroscopists at least should know about their
impact on an absorption measurement.
For suspensions with particles at a size d much smaller than the
wavelength (d ), i.e. up to some tens of nm for the wavelengths
used in UVVIS spectroscopy, Rayleigh scattering is observed. Protein
solutions are a typical example. The scattered light is classical dipole ra-
diation; the intensity varies with 1/4. Scattering for larger particles
(d b ), i.e. for cells and large nanoparticles sized up to some hundreds
of nm, is described by the Rayleigh-Gans-Debye model. This implies an
angular distribution of the scattering intensity different from Rayleigh
scattering, but also a 1/4 dependence. Scattering from larger particles
(d N or d ) is described by the Mie scattering model or by
Fraunhofer scattering, but these two are rare cases in bioanalytical
spectroscopy.
We thus expect on top of our absorption spectrum a
wavelength-dependent light scattering caused by Rayleigh or
Rayleigh-Gans-Debye scattering, thus an apparent absorption,
and we may call the sum of both terms extinction according to
the discussion above. Since absorption and apparent absorption are
essentially additive, they can be separated. Indeed they must be sep-
arated if quantitative absorption values are to be obtained from the
absorption spectrum. For this procedure, I see frequently quite futile
attempts. One possibility is to take values of the extinction outside
the range of the absorption band(s) and use these to calculate, with
a least-squares t, a 1/4 function as a new scattering baseline for
the entire extinction spectrum. If this is done carefully, a clean
absorption spectrum can be obtained by subtraction of this baseline
from the extinction spectrum.
Instead of post-experiment procedures, a careful choice of the ex-
perimental setup can reduce light scattering. Moving the detector
closer to the sample is a simple option. Doing this, the detector
area captures more of the scattered photons and the intensity loss
by scattering is reduced. Some top spectrometers have a second
separate sample compartment where the cuvettes are placed direct-
ly in front of the detector. This, however, bears the risk of also captur-
ing more uorescence photons as we discussed above. The sample-
detector distance is thus a compromise between reducing uores-
cence and reducing light scattering; this may be the choice between
a rock and a hard place.
A radical procedure to reduce light scattering for biological sam-
ples is refractive index matching. It makes use of the fact that the
scattering intensity of a suspension depends on the difference be-
tween the refractive index of the particle (the scatterer) and of
that of the solvent around. Adding soluble macromolecules, e.g.
long chain sugar molecules, to the solvent increases the refractive
index of the solution. Once this refractive index exactly matches
that of the particle, the suspension becomes clear scattering is
gone.
Finally, we consider the insufciency of the detection and signal
processing unit for absorbance measurements. We expect from a
 Editorial
photodetector high spectral sensitivity over the entire spectral range,
low dark noise, high linearity for the conversion of light intensity to
an electric signal, no drifts of the electric signal and no saturation for
high intensity. This is daydreaming, but nevertheless engineers have
done a good job.
In order to measure an absorbance of 1, detection and signal conver-
sion have to process two signals, I0 and I, that differ by a factor of 10 in
amplitude. For absorbances 2, 3 or 4, this factor is 100, 1000 and 10,000,
resp. These two signals must be amplied and digitized at a precision
that allows the calculation of I0/I and log (I0/I) with sufcient precision.
Digitization of an analog signal determines the resolution of the signal. If
a signal of 1 Volt is digitized at 10 bit resolution, it is resolved in 210 =
1024 equal discrete steps, each approx. 1 mV. Needless to say that if I
want to compare I0 and I that differ by a factor of 1.000 in amplitude,
the resolution of an analog-to-digital-converter must be quite a bit
higher.
Most signal processing units use analog-to-digital-converters that
are at 16 bit resolution; they divide the analog signal in 216 = 65.536
equal discrete steps. It is evident that even at that precision the digitiza-
tion of the smaller of both signals will be precision-limited. Higher
resolution analog-to-digital-converters are possible, but speed goes
down and prize goes up.
I0/I ratio formation is the next step. We need not be mathemati-
cians to see that the higher the absorbance and the smaller I is
compared to I0, the more we move towards a division by zero.
Small absolute differences in the measurement of I will cause large
changes in the result. This is another reason to keep the absorbance
low.
How do we notice any of these limitations in an absorbance mea-
surement? How do we detect violations of the Lambert-Beer law?
Very typically, the band proles change. They are typically Voigt pro-
les Gaussian distributions of Lorentz proles but become at at
the top and nally saturate in absorbance, no matter how concen-
trated the absorber is. Tops of the bands exhibit either strong noise
(no wonder if we force division by zero), or are completely at
when the spectrometer software determines that innity really
must be some arbitrary maximum value of, say, ve units. The linear
relation between concentration and absorbance gets lost. Instead of
determining quantitatively a concentration from a spectrum, you
might as well throw dice.
Fig. 1 illustrates this problem. Spectra of bovine serum albumin at in-
creasing concentrations were taken on one of our routine spectrome-
ters, neither badly adjusted on purpose, nor tuned to optimum. The
series of spectra shows band prole deformations starting at extinction
values above approx. 1.5. The inset shows the concentration as
Fig. 1. UV absorption spectra of bovine serum albumin (BSA) samples with increasing BSA
concentrations as indicated by the arrow. Note the band prole deformation at extinction
values above 1.5. Inset: The extinction values at 279 nm are plotted against the actual BSA
concentration demonstrating their linear relation up to extinction values around 1
(dashed line) and the deviation from this linearity above 1.5 (solid line).
967
determined from the absorbance of the aromatic amino acid side chains
at 279 nm vs. the real concentration.
What should the minimum precautions be?
In view of all these more or less hidden pitfalls that can mess up an
absorption measurement, a careful procedure should start with instru-
ment checks.
1. Take the time to have a look at the measuring beam of your
spectrometer and how it is placed with respect to the cuvette.
Simply set the monochromator to 500 nm, a wavelength where
eye sensitivity is highest, and check whether the beam is well cen-
tered in the cuvette and whether there might be spatial false light.
Check whether the sample lling volume is sufcient. If necessary,
readjust the sample position.
2. Run the spectrometer across the desired wavelength range without
any sample or reference cuvette, just air against air. Is the absor-
bance baseline thus obtained at or does it show offsets whenever
a lamp or order lter switch occurs? Adjustment of the optics may
be necessary.
3. Magnify this baseline to see the noise. What is the root mean square
level of this noise in terms of absorbance? For samples at low
extinction, this noise-equivalent absorbance will be your detection
limit.
4. Block the light path and measure a dark spectrum. The absor-
bance should max out everywhere; this will give you an idea
what your system does when insufcient light gets through the
sample.
5. Repeat the procedure in 1 with a pair of matched cuvettes both
lled with the same buffer/solvent. I know that this procedure is
rather old style, but having a pair of matched cuvettes, i.e. cuvettes
made from the same glass or quartz material and with pathlengths
as similar as possible, warrants a precise subtraction of buffer
absorbance. Refrain from using plastic cuvettes for quantitative
measurements if possible; their optical quality is not very high.
Again magnify the baseline thus obtained to see the noise. This
will tell you the usable spectral range.
6. Use the pair of matched cuvettes for the sample and the refer-
ence buffer/solvent to run a spectrum. If you do not have such
a pair, at least use the same cuvette for the measurement of the
background and the sample spectrum. If absorbance exceeds a
value of 12, then dilute and repeat. Aiming at a maximum
absorbance below 1 will prevent the uorescence problem
discussed above and guarantee a linear relationship between
absorbance and concentration. Lambert & Beer will bless you
for that.
7. If you are concerned that the lower concentration obtained by
dilution might have an impact on your sample, for example disag-
gregation of a protein, then use matched cuvettes with a shorter
path length instead of dilution. There are standard cuvettes from
quartz or glass at 1 cm or 5, 2, 1 mm and lower path lengths,
some even from plastic materials.
8. If a scattering background is observed, try to reduce scattering by
changing the geometry, i.e. move the sample and reference cuvettes
closer to the detector if possible.
9. Once the scattering is minimized on the experimental side, t a 1/4
function to the spectrum obtained, but exclude data points at and
immediately around the absorption band(s).
10. Subtraction of this 1/4 function will typically result in a rather
clean absorption spectrum that can be used for quantitative
evaluation.
All the pitfalls discussed here apply to standard absorption spectros-
copy in vitro. They may have a different impact depending on the
instrument used. Once spectroscopic experiments are performed outside
a well designed instrument, for example by using ber optics in situ or in
968 Editorial

biomedical spectroscopy in vivo, problems can become signicantly Further reading

worse.
In summary, it is easy to obtain clean, linear and quantitative absorp-
tion spectra if these rules are followed. I would not rely on post-
experimental processing of miserable data. Unfortunately, the instru-
ment manufacturers try to convince you that their instrument can
cope with these problems, but that is mostly a marketing claim. I
would rather wish they would build into their software a few program
lines that warn the user. At absorbance values up to about 1.5, the dis-
play should show a green light indicating that everything is OK. Above
W. Schmidt, Optical Spectroscopy in Chemistry and Life Sciences, Wiley VCH, 2005.
Glossary
Absorption: Decrease of light intensity of the measuring beam because molecules in the
sample undergo a transition from the ground state to an excited state
Extinction: Entire loss of light energy upon passing through a sample
Transmission: Ratio of light intensity before (I0) and after (I) the sample: (I/I0), often in
percent 100 (I/I0)

an absorbance of 1.5 to 2, an orange warning should ash up saying

you are about to leave the validity of Lambert-Beers Law, and above an
absorbance of 2, a warning light should appear in red and say you just
violated Lambert-Beers Law.