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

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Spectrochimica Acta Part A: Molecular and Biomolecular

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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 is typically called absorbance , A, and plotted vs. the wavelength.
to be necessary. We all use in our labs UVVIS spectrometers every day, Spectroscopy purists plot the molar absorption coefcient vs. the
either as routine instruments or for research, and thus should know the wavelength, and real hard core spectroscopists plot it vs. the wave-
rules. Yet in more than 10% of the manuscripts in my inbox I see crude number in cm 1.
violations of Lambert-Beer's law, either by wrong design of the experi- Please note that both transmission and absorption are dened on the
ment or because the prerequisites to apply the basic laws to measure basis of the ratio of two intensities, before and after the sample. No
transmission or absorption are not followed. Routine procedures bear matter what unit is used for I and I0, absorption and transmission are
the risk that potential pitfalls are not considered. dimensionless. For transmission, the value obtained is typically
Where are the problems? It starts with the fact that the terms multiplied by 100 and denoted as %T. For absorption, it is rather a bad
absorption and extinction are very often used synonymously and habit if spectroscopists use absorbance units, AU, or OD for optical
used as labels at the ordinate of the spectrum plots. I wish we would be density units.
more precise here. Absorption is dened as the process whereby the While these are merely semantic problems or badly dened physical
light intensity from the measuring beam is diminished because molecules magnitudes, the pitfalls on the technical side are more relevant. When
in the sample undergo a transition from the ground state (usually the we learnt about the use of the Lambert-Beer law in spectroscopy, we
singlet state S0 for molecules at room temperature) to an excited state also learnt that it is only strictly valid if some fundamental conditions
S1, S2, or higher. Extinction refers to the entire loss of light energy are fullled. The most relevant are:
upon passing through the sample. It includes absorption, but also light
scattering and reection, processes that have nothing to do with the strictly monochromatic measuring light;
absorption process. The extinction is thus equal to or higher than the homogeneous distribution of the molecules in the sample;
absorption. passage of the complete measuring beam through the sample;
The numerical value obtained in a UVVIS spectroscopy experiment absence of light scattering and of photochemical reactions in the
by application of Lambert-Beer's law (historically more correct: the sample;
Bouguer-Lambert-Beer Law): no re-emission of the absorbed light by uorescence;
an ideal detection and processing of the intensity values I0 and I.
A logT log cd
Real life is not ideal, and so is not absorption spectroscopy. The rst
T: transmission; condition strictly monochromatic light is already hard to guarantee.
I0, I: intensity of the measuring beam before/after passing through Our routine spectrometers typically use a tungsten-iodine light source
the sample; for the near-UV to near IR spectral range (e.g. 3801000 nm) and a
: molar absorption coefcient; deuterium discharge lamp for the UV from about 200 to 380 nm. A ip
c: concentration; mirror is used to switch between the lamps. For a perfectly adjusted
d: path length of the measuring beam in the sample. optics, the absorbance values of a sample before and after ipping this
mirror should be identical, but most spectra (including the ones I see
Corresponding author. in incoming manuscripts) show an offset. In order to obtain monochro-
E-mail address: (W. Mntele). matic light (a euphemism, because strictly monochromatic would
1386-1425/ 2016 Elsevier B.V. All rights reserved.
966 Editorial

mean that the intensity is zero), most instruments use a grating are typical for rst-year students, but I have seen this for experi-
monochromator that is moved by a stepper motor. The spectral enced senior scientists in research labs. The so-called microcuvettes
bandwidth of the output of this monochromator is a compromise be- that are popular because they require only small sample volumes are
tween intensity and spectral bandwidth set by the mechanical width prone to this problem, since they have only a narrow slit for the sam-
of the monochromator output slit. Since 12 nm spectral bandwidth ple with glass or plastic walls left and right. The slightest shift of
can be easily obtained with still sufcient light intensity, this is not real- these cuvettes causes the measuring beam to pass partly through
ly a problem. The pitfalls come from the fact that any grating monochro- the glass walls. The solution can be as trivial as the problem: tale a
mator uses the grating at a certain diffraction order: rst, second, third black felt pen and paint the glass walls left and right from the sample
and so on. The interference conditions of a grating state that at any out- part until they are intransparent. There are also blackened cuvettes
put wavelength the grating is set, there is also intensity at higher or- available; while they are more expensive, their use should be much
ders, i.e. 2, 3 and so forth, albeit at much lower intensity. This preferred in particular for very small sample volumes.
means that if your monochromator is set at blue-violet light at Light scattering in the sample is also a false light problem. The ideal
400 nm, the measuring beam also contains light at 800 nm. Just imagine sample in the sense of the Lambert-Beer law is a homogeneous solution
that if your sample absorbs strongly at 400 nm but is transparent at without light scattering. Many chemical, biochemical or biological
800 nm; the detector will not be able to correctly measure the residual samples are suspensions or textured structures that exhibit light
intensity at 400 nm because of the fraction of light at 800 nm detected. scattering. It is not always necessary to describe light scattering
Manufacturers of spectrometers cope with this physics of diffraction by quantitatively, but spectroscopists at least should know about their
placing so-called order lters in the beam after the monochromator impact on an absorption measurement.
that let the desired light pass and block or at least reduce the higher For suspensions with particles at a size d much smaller than the
order light intensity. Nevertheless, there are traces of unwanted light wavelength (d ), i.e. up to some tens of nm for the wavelengths
in the measuring beam that limit the application of the Lambert-Beer used in UVVIS spectroscopy, Rayleigh scattering is observed. Protein
law. This phenomenon is frequently called spectral false light, in con- solutions are a typical example. The scattered light is classical dipole ra-
trast to spatial false light that we will deal with below. diation; the intensity varies with 1/4. Scattering for larger particles
Another source of spectral false light is stray light in the monochroma- (d b ), i.e. for cells and large nanoparticles sized up to some hundreds
tor. This means that a small but noticeable fraction of the incoming white of nm, is described by the Rayleigh-Gans-Debye model. This implies an
light will also appear at the output. For a well-designed monochromator, angular distribution of the scattering intensity different from Rayleigh
stray light is on the order of 103 to 105 of the incoming light intensity. scattering, but also a 1/4 dependence. Scattering from larger particles
Stray light is equally sensed by the photodetector and also limits the min- (d N or d ) is described by the Mie scattering model or by
imum measurable intensity or the maximum of measurable Fraunhofer scattering, but these two are rare cases in bioanalytical
absorbance. spectroscopy.
A third source of spectral false light may occur if the sample is strongly We thus expect on top of our absorption spectrum a
uorescent. Let us assume that the sample has a peak absorbance of 2 wavelength-dependent light scattering caused by Rayleigh or
which means that only 1% of the measuring light is passing at this wave- Rayleigh-Gans-Debye scattering, thus an apparent absorption,
length. If the uorescence quantum efciency of the sample is high, a high and we may call the sum of both terms extinction according to
fraction of the absorbed photons (99% in our example) is re-emitted as the discussion above. Since absorption and apparent absorption are
uorescence. Again, the photodetector does not care whether it sees the essentially additive, they can be separated. Indeed they must be sep-
residual intensity I (1% in our example) of the measuring light or the in- arated if quantitative absorption values are to be obtained from the
tensity caused by the re-emitted photons at a wavelength shifted to the absorption spectrum. For this procedure, I see frequently quite futile
red. If the detector is more sensitive for the uorescence wavelength, attempts. One possibility is to take values of the extinction outside
the uorescence intensity can appear even higher than the transmitted the range of the absorption band(s) and use these to calculate, with
intensity. The precise estimation of this error source requires knowledge a least-squares t, a 1/4 function as a new scattering baseline for
about the geometry of the sample, the optics and the detector. the entire extinction spectrum. If this is done carefully, a clean
Molecules may diffuse and rotate while the molecule is in the excited absorption spectrum can be obtained by subtraction of this baseline
state; uorescence emission is thus not in the direction of the measuring from the extinction spectrum.
beam, but rather isotropic. Instead of post-experiment procedures, a careful choice of the ex-
There are means to get rid of the spectral false light caused by perimental setup can reduce light scattering. Moving the detector
sample uorescence. The best solution is a second monochromator closer to the sample is a simple option. Doing this, the detector
between sample and detector, set at equal spectral resolution and area captures more of the scattered photons and the intensity loss
moved synchronously with the rst monochromator. This guaran- by scattering is reduced. Some top spectrometers have a second
tees that only the measuring light passes from the sample to the separate sample compartment where the cuvettes are placed direct-
detector. This luxury solution solves the problem caused by uores- ly in front of the detector. This, however, bears the risk of also captur-
cent samples and the problem caused by stray light, but not the ing more uorescence photons as we discussed above. The sample-
higher order problem, unless order lters are included, too. The detector distance is thus a compromise between reducing uores-
cheaper solution is to increase the distance between the sample cence and reducing light scattering; this may be the choice between
and the detector and to use a parallel beam of light for the absorption a rock and a hard place.
measurement. If so, the transmitted measuring light I is recorded A radical procedure to reduce light scattering for biological sam-
independent from distance r, while the intensity of the uorescence ples is refractive index matching. It makes use of the fact that the
light will decrease approximately with 1/r2 because it is emitted scattering intensity of a suspension depends on the difference be-
almost isotropically. An even cheaper solution is dilution of the tween the refractive index of the particle (the scatterer) and of
sample. As you dilute, the transmitted intensity I will increase that of the solvent around. Adding soluble macromolecules, e.g.
while the emitted uorescence intensity will decrease. long chain sugar molecules, to the solvent increases the refractive
Spatial false light has many origins. There are trivial ones, such as index of the solution. Once this refractive index exactly matches
incompletely lled cuvettes where the measuring beam partly that of the particle, the suspension becomes clear scattering is
passes above the sample, and this fraction reaches the detector unat- gone.
tenuated. There are badly adjusted cuvette holders that let part of Finally, we consider the insufciency of the detection and signal
the light pass sideways. You may think that I report problems that processing unit for absorbance measurements. We expect from a
Editorial 967

photodetector high spectral sensitivity over the entire spectral range, determined from the absorbance of the aromatic amino acid side chains
low dark noise, high linearity for the conversion of light intensity to at 279 nm vs. the real concentration.
an electric signal, no drifts of the electric signal and no saturation for What should the minimum precautions be?
high intensity. This is daydreaming, but nevertheless engineers have
done a good job. In view of all these more or less hidden pitfalls that can mess up an
In order to measure an absorbance of 1, detection and signal conver- absorption measurement, a careful procedure should start with instru-
sion have to process two signals, I0 and I, that differ by a factor of 10 in ment checks.
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 1. Take the time to have a look at the measuring beam of your
that allows the calculation of I0/I and log (I0/I) with sufcient precision. spectrometer and how it is placed with respect to the cuvette.
Digitization of an analog signal determines the resolution of the signal. If Simply set the monochromator to 500 nm, a wavelength where
a signal of 1 Volt is digitized at 10 bit resolution, it is resolved in 210 = eye sensitivity is highest, and check whether the beam is well cen-
1024 equal discrete steps, each approx. 1 mV. Needless to say that if I tered in the cuvette and whether there might be spatial false light.
want to compare I0 and I that differ by a factor of 1.000 in amplitude, Check whether the sample lling volume is sufcient. If necessary,
the resolution of an analog-to-digital-converter must be quite a bit readjust the sample position.
higher. 2. Run the spectrometer across the desired wavelength range without
Most signal processing units use analog-to-digital-converters that any sample or reference cuvette, just air against air. Is the absor-
are at 16 bit resolution; they divide the analog signal in 216 = 65.536 bance baseline thus obtained at or does it show offsets whenever
equal discrete steps. It is evident that even at that precision the digitiza- a lamp or order lter switch occurs? Adjustment of the optics may
tion of the smaller of both signals will be precision-limited. Higher be necessary.
resolution analog-to-digital-converters are possible, but speed goes 3. Magnify this baseline to see the noise. What is the root mean square
down and prize goes up. level of this noise in terms of absorbance? For samples at low
I0/I ratio formation is the next step. We need not be mathemati- extinction, this noise-equivalent absorbance will be your detection
cians to see that the higher the absorbance and the smaller I is limit.
compared to I0, the more we move towards a division by zero. 4. Block the light path and measure a dark spectrum. The absor-
Small absolute differences in the measurement of I will cause large bance should max out everywhere; this will give you an idea
changes in the result. This is another reason to keep the absorbance what your system does when insufcient light gets through the
low. sample.
How do we notice any of these limitations in an absorbance mea- 5. Repeat the procedure in 1 with a pair of matched cuvettes both
surement? How do we detect violations of the Lambert-Beer law? lled with the same buffer/solvent. I know that this procedure is
Very typically, the band proles change. They are typically Voigt pro- rather old style, but having a pair of matched cuvettes, i.e. cuvettes
les Gaussian distributions of Lorentz proles but become at at made from the same glass or quartz material and with pathlengths
the top and nally saturate in absorbance, no matter how concen- as similar as possible, warrants a precise subtraction of buffer
trated the absorber is. Tops of the bands exhibit either strong noise absorbance. Refrain from using plastic cuvettes for quantitative
(no wonder if we force division by zero), or are completely at measurements if possible; their optical quality is not very high.
when the spectrometer software determines that innity really Again magnify the baseline thus obtained to see the noise. This
must be some arbitrary maximum value of, say, ve units. The linear will tell you the usable spectral range.
relation between concentration and absorbance gets lost. Instead of 6. Use the pair of matched cuvettes for the sample and the refer-
determining quantitatively a concentration from a spectrum, you ence buffer/solvent to run a spectrum. If you do not have such
might as well throw dice. a pair, at least use the same cuvette for the measurement of the
Fig. 1 illustrates this problem. Spectra of bovine serum albumin at in- background and the sample spectrum. If absorbance exceeds a
creasing concentrations were taken on one of our routine spectrome- value of 12, then dilute and repeat. Aiming at a maximum
ters, neither badly adjusted on purpose, nor tuned to optimum. The absorbance below 1 will prevent the uorescence problem
series of spectra shows band prole deformations starting at extinction discussed above and guarantee a linear relationship between
values above approx. 1.5. The inset shows the concentration as 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

Fig. 1. UV absorption spectra of bovine serum albumin (BSA) samples with increasing BSA All the pitfalls discussed here apply to standard absorption spectros-
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
copy in vitro. They may have a different impact depending on the
concentration demonstrating their linear relation up to extinction values around 1 instrument used. Once spectroscopic experiments are performed outside
(dashed line) and the deviation from this linearity above 1.5 (solid line). 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

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