specwochimla Ata Part A: Molecular and Biomolecular Spectroscopy 173 (2017) 965-968 ELSEVIER CContontsIsts availble at ScienceDirect Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.clsevier.com/locate/saa Editorial UV-VIS absorption spectroscopy: Lambert-Beer reloaded Werner Mantele *, Erhan Deniz nse ir py ohan Wolo Corte Univers rani cn Ma, Maton ave Se 1, 0-908 Panto Mal Cemany ‘alae online 2 September 2016 ‘UVAMS absorption specroscopys used in almost every spectroscopy laboratory fr routine analysis research, All spectroscopist ely on the Lambert-Beer Law bus many of hem are less ware fits mations. This tutorial Aiscusses typical problems in routine spectroscopy that come along with tecinicallinitations or careless selection of experimental parameters. Simple rules are provided to avid these problems. ‘© 2016 Elevier Allright reserved ‘A tutorial on UV-Vis spectroscopy? At fist sight, this does not seem tobe necessary, We all use in our labs UV-VIS spectrometers every day, either as routine instruments or for research, and thus snould 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 pitfals are not considered Where are the problems? It starts with the fact that the terms absorption” and “extinction” are very often used synonymously and sed as labels atthe ordinate ofthe spectrum plots. wish we would be more precise here. “Absorption” i defined as the process whereby the light intensity from the measuring beamis diminished because molecules inthe sample undergo a transition from the ground state (usually the singlet state Sp for molecules at room temperature) to an excited state 5, Sp oF higher. “Extinetion” refers tothe entire loss of light energy ‘upon passing through the sample. I includes absorption, but aso light scattering and reflection, processes that have nothing to clo with the Absorption process. The extinction is thus equal to or higher than the absorption The numerical value obtained in a UV-VIS spectroscopy experiment by application of Lambert-Beer's law (historically more correct: the Bouguer-Lambert-Beer Law) Jo. intensity of the measuring beam before/after passing through the sample ‘: molar absorption coefficient; concentration: dt pathlength ofthe measuring beam inthe sample TT Carespanaing autor ‘alors matsteleebophysiuni-tanlct de (W. Mace) prog t01065}500201609057 385-2500 2015 Hae BV light seve {s typically called “absorbance”, A, and plotted vs. the wavelength Spectroscopy puists plot the molar absorption coefficient « vs. the wavelength, and real hard core spectroscopists plot it vs. the wave- ‘number in cm! Please note that both transmission and absorption are defined on the basis of the ratio of two intensities, before and after the sample, No ‘matter what unit is used for I and ly, absorption and transmission are dimensionless. For transmission, the value obtained is typically ‘multiplied by 100 and denoted as %7. For absorption, itis rather a bad habit if spectroscopists use “absorbance units", AU, or OD for “optical density units" ‘While these are merely semantic problems or badly defined physical ‘magnitudes, the pitfalls on the technical side are more relevant, When ‘we learnt about the use ofthe Larabert-Beer law in spectroscopy. We als learnt that its only strictly valid if some fundamental conditions are fulfilled, The most relevant ar: + strictly monochromatic measuring light: + homageneous distribution of the molecules in the sample; + passage ofthe complete measuring beam through the sample; + absence of light scattering and of photochemical reactions in the sample; + no re-emission ofthe absorbed light by fluorescence: + an ideal detection and processing ofthe intensity values Ip and Real lifes not eal, and so isnot absorption spectroscopy. The fist condition - strictly monochromatic light ~ is already hard to guarantee. (Our routine spectrometers typically use a tungster-iodine ight source for the near-UV to neat IR spectral range (e.g 380-1000 nm) and a deuterium discharge lamp forthe UV from about 200 to 380 nm. A flip mirror is used to switch between the lamps. Fora perfectly adjusted ‘optics, the absorbance values of a sample before and after flipping this :mirtor should be identical, But most spectra (including the ones I see inincoming manuscripts) show an offset In order to obtain “monochro- matic light" (a euphemism, because strietly monochromatic would6 ao mean that the intensity is zero), most instruments use a grating ‘monochromator that is moved by a stepper motor. The spectral bandwidth ofthe output ofthis monochromator is a compromise be- tween intensity and spectral bandwidth set by the mechanical width of the monochromator output slit Since 1-2 nm spectral bandwidth can be easily obtained with stil sufficient light intensity, this isnot real Iya probler. The pitfalls come from the fact that any grating monochro- ‘mator uses the grating ata certain diffraction order: first, second, third and so on. The interference conditions ofa grating state that at any out- put wavelength \ the grating i set, there is also intensity at higher or- ders, ie. 2. 34 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 nun 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 at400 nm because of the fraction of light a “Manufacturers of spectrometers cope with this physics placing so-called “order iter in the beam after the monochromator that let the desired light pass and block or atleast reduce the higher order light intensity, Nevertheless, there are traces of unwanted light in the measuring beam that limit the application ofthe Lambert-Beer lave This phenomenon is frequent called “spectral false light trast to “spatial false ight” that we will deal with below. ‘Another source of spectral false lights stray ight in the monochroma- tor. This means that a small bu noticeable fraction ofthe incoming white light will also appear tthe output For a well-designed monochromator, stray light i on the order of 10 to 10-* ofthe incoming light intensity Straylight is equally sensed by the photodetector and als limits the min- imum measurable intensity — or the maximum of measurable absorbance, ‘third source of spectral false light may occurifthe sampleis strongly fluorescent, Let us assume thatthe sample has a peak absorbance of 2 Which means tha only 1% of the measuring light is passing at this wave- length ithe Nuorescence quantum efficiency of the sample ishigh, high fraction ofthe absorbed photons (99% in our example) is re-emitted as fluorescence. Again, the photodetector does not care whether it ses the residual intensity 1 (1% in our example) of the measuring light or the in- tensity caused by the remitted photons at a wavelength shifted tothe ted. Ifthe detector is mote sensitive forthe fluorescence wavelength the fluorescence intensity can appear even higher than the transmitted intensity. The precise estimation ofthis 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; fuorescence emission is thus not inthe direction of the measuring beam, but rather isotropic There are means to get rid of the spectral false light caused by sample fluorescence. The best solution is a second monochromator between sample and detector, set at equal spectral resolution and ‘moved synchronously with the first monochromator. This guaran- tees that anly the measuring light passes from the sample to the detector. Ths luxury solution solves the problem caused by fluores- cent samples and the problem caused by stray light, but not the higher order problem, unless order filters 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 fr the absorption ‘measurement. Ifso, the transmitted measuring light lis recorded independent from distance r, while the intensity ofthe fluorescence light will decrease approximately with 1/72 because itis emitted almost isotropically. An even cheaper solution is dilution of the sample. As you dilute, the transmitted intensity 1 will increase ‘while the emitted fluorescence intensity will decrease. Spatial false light has many origins. There ae trivial ones, such as, incompletely filled cuvettes where the measuring beam partly passes above the sample, and this fraction reaches the detector unat- enuated. There are badly adjusted cuvette holders that let part of the light pass sideways. You may think that I report problems that are typical for first-year students, but I have seen this for experi- tenced senior scientists in research labs, The so-called microcuvettes that are popular because they require only small sample volumes are prone tothis problem, since they have only anarrow slit forthe sam- ple with glass ar plastic wall let and right. The slightest shift of these cuvettes causes the measuring beam to pass party through the glass walls. The solution can be as trivial as the problem: tale a black felt pen and paintthe glass walls left and right from the sample part until they are intranspareat. 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 inthe sample is also a als light problem. The ideal sample in the sense ofthe Lammbert-Beet law isa 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 atleast should know about their impact on an absorption measurement. For suspensions with particles ata size d much smaller than the wavelength (d « d), ie, up to some tens of nm forthe wavelengths used in UV-VIS spectroscopy, Rayleigh scattering is observed. Protein solutions are atypical example, The scattered light i classical dipole ra- ation; the intensity varies with 1/M*, Scattering for larger particles (dN), te. for cell and large nanoparticles sized up to some hundreds of nm, is described by te Rayleigh-Gans-Debye model. This implies an angular distribution of the scattering intensity diferent from Rayleigh scattering but also a 1/\* dependence, Sattering fram larger particles (d > or d > A) is described by the Mie scattering mode! 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 procedute, I see frequently quite futile attempts. One possibility is to take values of the extinction outside the range of the absorption bancl(s) and use these to calculate, with a least-squares fit, a 1/A* 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 fom the “extinction” spectrum. Instead of past-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 dicect- lyin front ofthe detector. This, however, bears the risk of also capcur- ing more fluorescence photons as we discussed above. The sample- etector distance is thus a compromise between reducing fluores- cence and reducing light scattering; this may be the choice "between rock and a hard place” ‘A tadical procedure to reduce light scattering for biological sam- ples is “refractive index matching". It makes use ofthe fact that the scattering intensity of suspension depends on the diflerence be- tween the refractive index of the particle (the "scatterer”) and of that of the solvent around. Adding soluble macromolecules, eg 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 insufficiency ofthe detection and signal processing unit for absorbance measurements, We expect from aton sr photodetector high spectral sensitivity over the entire spectral range, low dark noise, high linearity forthe conversion of light intensity to ‘an electri signal, no drifts ofthe electre signal and no saturation for high intensity. This is daydreaming, but nevertheless engineers have done a good jo. In order to measure an absorbance of 1, detection and signal conver= sion have to process two signals, I and I that diffe by a factor of 10in amplitude. For absorbances 2,3 or 4, this actor is 100, 1000 and 10,000, resp, These (wo signals must be amplified and digitized at a precision hat allows the calculation of ly and log (ly) with sufficient precision, Digitization ofan analog signal determines the resolution ofthe signal. IF a signal of 1 Volt is digitized at 10 bit resolution, iis resolved in 2"° = 1024 equal discrete steps, each approx. 1 ml. Needless to say that iT ‘want to compare lp and | that eifer by a factor of 1,000 in amplitude, the resolution of an analog-to-digital-converter must be quite abit higher. ‘Most signal processing units use analog-to-digital-converters that are at 16 bit esolution; they divide the analog signal in 2"° = 65.536 fequal discrete steps. tis evident that even at that precision the eiitiza- tion of the smaller of both signals will be precsion-limited. Higher resolution analog-to-digital-converters are possible, but speed goes down ane prize goes up. Jo(l rato formation is the next step. We need not be mathemati- cians to see that the higher the absorbance and the smaller | is compared to fp, 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 ofthese limitations in an absorbance mea- surement? How do we detect violations of the Lambert-Beer law? ‘Very typically, the band profiles change. They are typically Voigt pro- files - Gaussian distributions of Lorentz profiles - but become fat at he top and finally saturate in absorbance, no matter how concen rated the absorber is. Tops ofthe bands exhibit either strong noise (no wonder if we force division by zero), or are completely flat When the spectrometer software determines that “infinity” really :must be some arbitrary maximum value of, say, five units. The linear relation between concentration and absorbance gets lost. Instead of determining quantitatively a concentration from a spectrum, you right as well throw dice, ‘ig. illustrates this problem Spectra of bovine serum albumin atin- creasing concentrations were taken on one of our routine spectrome- ters, nether bacly adjusted on purpose, nor tuned to optimum, The seties of spectra shows band profile deformations starting at extinction values above approx. 1.5, The inset shows the concentration as 2,0 18 012345678 10 ‘cone, [mg/ml extinction E 05 0 ; 260 280 300 320 340 360 380 wavelength 2. [nm] Fig 1. abepton seca fie serum abumin ASA) simples wth nese BSA concentrations inceateby te aow. Nae te bane profile eration at exten ‘aloes above Ins Teen aes 3279 amare pte ine he 3 398 fonction gemonstrting the lina relation upto extinction values ouné 1 {eishe ne} an he deviation rm is inert above 1 (0 ne) determined from the absorbance ofthe aromatic amino acd side chains at 279 nm vs the real concentration. What should the minimum precautions be? In view ofall 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 tye sensitivity is highest, and check whether the beam is well cen= tered in the cuvette and whether there might be spatial false ight Check whether the sample filing volume is sufiient-Ifnecessary, readjust the sample postion. 2, Runthe spectrometer across the desired wavelength range without any sample or reference cuvette, just alr against ai. Is the absor- bance baseline thus obtained lat or does it show offsets whenever lamp or order filter switch occurs? Adjustment ofthe optics may be necessary, 5, 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 éark spectrum. The absor= bance should max out everywhere; this will give you an idea ‘what your system does when insufficient light gets through the sample 5, Repeat the procedure in 1 with a pair of matched cuvettes both filled with the same buffer/solvent. I know that this procedure is rather old style, but having a pair of matched cuvettes. cuvettes ‘mac 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 wil tell you the usable spectral range. 6, Use the pair of matched cuvettes for the sample and the refer- tence buffer/solvent to run a spectrum, If you do not have such 4 pair, atleast use the same cuvette forthe measurement of the background and the sample spectrum. [fabsorbance exceeds a value of 1-2, then dilute and repeat. Aiming at a maximum absorbance below 1 will prevent the fluorescence problem discussed above and guarantee a linear relationship between absorbance and concentration. Lambert & Beer will bless you for that. 7. Ifyou are concerned that the lower concentration obtained by dliluton might have an impact on your sample, for example disag- sregation 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, Ifa scattering background is observed, try to reduce scattering by changing the geometry, ie. move the sample and reference cuvettes closer to the detector if possible, 8, Once the scattering is minimized on the experimental side, ita 1/A* function o the spectrum obtained, but exclude data points at and immediately around the absorption band(s). 10, Subtraction ofthis 1/9 function will typically result ina rather Clean absorption spectrum that can be used for quantitative ‘evaluation Alle pitfalls ciscussed here apply to standard absorption spectros- copy in vitro. They may have a different impact depending on the instrument used. Once spectroscopic experiments ae performed outside a well designed instrument, fo example by using fiber optics in sy or in8 aon biomedical spectroscopy in vivo, problems can become significantly In summary, itis easy to obtain clean, linear and quantitative absoxp- tion spectra if these rules are followed. | 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. 1 ‘would rather wish they would build into ther 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 an absorbance of 1.5 to 2, an orange warning should flash up saying “you are about to lave the validity of Lamber-Beers Law’, and above an absorbance of 2, a warning light should appear in ted and say "you just Violated Lamnber-Beers Law” Further reading cl pecooscopy in Chemistry and Licences Wey VCH. 2005, ‘Absorption, Deas fight nensy ofthe measuring bean because molecules ate ‘Simpl undergo tansion Hao the proud sate oan excite sae tin ae oss fight ener pon passing hoop a sar Tronsmison: Fate of ig intensty before) ad ater (the sample (i). oem in cet 100% ts)