In Absorption UV-Visible spectroscopy, the absorption is proportional to concentration according to Beer Lambert's law. However, this is not followed in higher and low concentration of a particular metallic solution.
Go through this page which explains the deviations and limitations of Beer Lamber Law.
http://pharmaxchange.info/press/2012/05/ultraviolet-visible-uv-vis-spectroscopy-%E2%80%93-limitations-and-deviations-of-beer-lambert-law/
I Guess, A part of the absorbed radiation will be emitted back by self quencing (collision), so we will get eranius result on higher concentration. At lower concentration the amount of radiation absorbed will be undetectable.
Go through this page which explains the deviations and limitations of Beer Lamber Law.
http://pharmaxchange.info/press/2012/05/ultraviolet-visible-uv-vis-spectroscopy-%E2%80%93-limitations-and-deviations-of-beer-lambert-law/
What kind of deviation do you observe at low concentrations? And how low are "low concentrations" for you? Do you actually mean a solution of a metal salt or a dispersion of metallic nanoparticles.
Lambert Beer law at high concentrations cannot give good correlations because when the absorbance is higher than 1, it is absorbed all light. Under these conditions the absornabance is not coorrect (see definition of absorbance).
At low concentrations, lower than 0.04 the measured has to much error, this leads to important precision of the absorbance measurement.
Lambert Beer law at high concentrations cannot give good correlations because when the absorbance is higher than 1, it is absorbed all light. Under these conditions the absornabance is not coorrect (see definition of absorbance).
At low concentrations, lower than 0.04 the measured has to much error, this leads to important precision of the absorbance measurement.
Limitations / Deviations* :
It is capable of describing absorption behavior of solutions containing relatively low amounts of solutes dissolved in it (10mM), the analyte begins to behave differently :
[i]Due to interactions with the solvent and other solute molecules.
[ii]Solute molecules can cause different charge distribution on their neighboring species.
[iii]Since UV-visible absorption is an electronic phenomenon, high concentrations would possibly result in a shift in the absorption wavelength of the analyte.
[iv] At times, even electrolyte concentrations (such as those present in buffers) play an important role in altering the charge distributions and affecting UV-visible absorbance.
[v] Some large ions or molecules show deviations even at very low concentrations. For example. methylene blue absorptivity at 436 nm fails to observe Beer Lambert law even at concentrations as low as 10μM.
[vi]High analyte concentrations MAY alter the refractive index (η) of the solution which in turn could affect the absorbance obtained. If addition of solute causes a significant change in the refractive index of the solution, then correct form of Beer Lambert is as:
A=εbc(ɳ^2+2)^2
This correction is normally not required below10mM concentrations .
* Given time/ space, one may add one/ two more deviation to this list.
Usually, when you reach the limits of Beers law, you encounter problems with your instrument sensitiity. Your measures values are I and I_0.
At very low concentrations, the difference of these values gets so small, that their ratio approaches 1, which means the observed transmittance approaches 1.
For very high concentrations, the ratio I / I_0 approaches 0, so hardly any light is transmitted.
The ratio of two very much identical large numbers (too low concentrations) or of a large and a very small number (low concentrations) is very much affected by noise and detection sensitivity.
Additionally, there are problems of self-quenching, inner filter effect, at high concentrations that introduce deviations. The reply by Manohar Sehgal gives a good overview of the limitations.
As pointed out at low concentration the problem is accuracy and taking into consideration the real reference sample, i.e. subtracting the extinction related to the cell and continuous phase.
At high extinction values, e.g. 2 and higher there is a very remarkable problem. Lambert Beer law holds for a single wavelength only. Scattering will cause an overlay with light of other wavelength leading to a drastic distortion of the spectra. There was a very detailed work of W. A. P. LUCK in relation to these phenomena more than 50 years ago.
In continuation of my above given answer, as promised, I, humbly, mention two more deviations as follows :
[I]Depending upon concentration, the analyte molecules may undergo association, dissociation and interaction with the solvent to produce a product with different absorption characteristics. For example, phenol red undergoes a resonance transformation when moving from the acidic form (yellow) to the basic form (red). Due to this resonance, the electron distribution of the bonds of molecule changes with the pH of the solvent in which it is dissolved.
Since UV-visible is electron-related phenomenon, the absorption spectrum of the sample changes with the change in pH of the solvent.
[II] Lastly, some molecule of the same substance, undergong association and the other dissociation will have two molar absorptivities ε/ and ε// at wave lengths λ/ and λ//. The absorbance (A) for such a species can be calculated as:
A=log(1/+I//)/ (I/ .10^- ε/ bc +I//.10^- ε// bc )
When the molar absorptivities are the same at both wavelengths ( ε/ = ε//) , the relationship between absorbance and concentration follows Beer-Lambert law to obtain a straight line. However, as the difference between ε/ and in ε// in creases, the deviations from linearity also increases.
it is impossible to give good answer if you do not know the chemical nature of analytical system because besides physical reasons there are many chemical ones exist.
Lambert-Beer will always be obeyed.
If you see deviations in your experiments:
-it is related to your experimental accuracy or error (don't know the setup, but reflection losses at the air/cuvette/sample interfaces)
-your assumptions are not adequate like only the solute absorbing (absorption of the solvent may become important at low concentrations)
-or you solute undergoes some changes (e.g. aggregation,...) which will affect the extinction coefficient
Addition: an absorbance of 1 does not mean that all light is absorbed, but 90% is absorbed
Look at page 59 of Principles of Fluorescence Spectroscopy, 3rd Ed. by JR Lakowicz (Springer 2006)
In UV-Visible spectroscopy, absorption is due to presence of chromophore and auxochromes. Each color has its lowest and highest limitations of wavelength absorption range. say pink color Faint to dark out highest dark color intensity is obtained no further change is possible and it vice vars a for lower concentration.
hence with in specific concentration range only Beer Lambert's law is obayed.
The problem is a non-linearity in aborbance D which is in fact quadratic or even cubic equation. A part is described in our paper (Barankova et al. 2016, RSE) which can be donwloaded from my RG page
Petr
what is the meaning of
"absorption cross-sections of individual molecules are in alignment?" and where is your reference
The nature of the transition being observed can also be very important. For example, d-d transitions are within tansition metal complexes and therefore not modified by changing the solvent or additions of non-interacting ions. However, the peak of the uv spectrum of the iodide ion depends on the solvent because the transition being recorded is a CTTS transition (Charge Transfer To Solven). Thus by adding say sodium chloride to an aqueous solution of NaI or KI the change in the ionic atmosphere around iodide will cause a shift in its peak position. Such small shifts are usually linear with increasing concentration of the added chloride or similar ions.
1. Trevor R. Griffiths and Martyn C. R. Symons
Ionic interactions in solutions of electrolytes as studied by ultraviolet spectroscopy
Molecular Physics, 3, 90‑102, 1960.
2. Trevor R. Griffiths and Martyn C. R. Symons
Solvation Spectra Part 3. Further studies of the effect of environmental changes on the ultraviolet spectrum of iodide
Transactions of the Faraday Society, 56, 1125‑1136, 1960.
I think this link is very useful,
http://www.instanano.com/characterization/theoretical/concentration-calculation-from-uv-vis-absorbance/
The solution is obeyed by this law at two conditions: 1. the light is monochromatic; this condition maintaines by using monochromatic wavelength in spectrophotometer.
2. the concentration of absorbing species in solution should not change spontaneously. This condition very often not obeys. See reasons below.
Low concentration of absorbing species (metal+ligand): in this case it can be dissociation of unstable complex, hydrolysis (solvolysis) of components, or side reactions with buffer components or other additives (contaminants) as well as change the pH of solution or tautomerism. All this factors change the concentration (speciation) of absorbing species.
At high concentration the reasons for the deviations may be a formatiom of complex with another composition or dimerization (aggregation) of reagent (ligand) or complex, high ionic strength can also influence on the activity of species or their electronic state, or will have dehydration and medium polarity effects.
To avoid these confusing factors you should know the composition of your solution and foresee possible interaction inside of them.
Even if Beer's Law were perfectly obeyed which it never is if only because of chromophore "shadowing" it would not be apparent because of imposed instrumental artifacts. Sorry . . .
For high concentrations, or long optical paths, more than 1 absorption emission event can occur resulting in a broadening in the absorption peak and a shift away from Beer's Law. This occurs for a photon in the atmosphere, and also occurs in chloroplast in plants where this behavior is used to improve the efficiency of photosynthesis through the capture of more wavelengths of light.
If one chromophore give you a certain absorbance value which doubles when you add a second chromophore, what happens to the absorbance level when the second chromophore passes behind the first?????
@Harry Harlow
can you give an example for the atmosphere: the optical density of most substances is low
We have published a paper by Naus et al. (Photosynthesis Research, 2018, in press) on this topic. The paper can be dowloaded on the PR site.
This thread is somewhat symptomatic for spectroscopy, where optical, physical and chemical reasons why and when Beer-Lambert fails are nearly inextricable intermixed and intertwined, as are Maxwell-confom and non-conform concepts.
Let's make things very simple first and investigate the optical properties of a diluted solution (perfect homogeneously mixed) with a transparent solvent. From an optical, i.e. Maxwell-conform view of the problem, the Beer-Lambert law describes propagation inside (and not transmission through) a medium, i.e. inside the solution. Once there are interfaces and there are, since liquids need cuvettes to be measured, the Beer-Lambert law is no longer obeyed and the optical model becomes much more complicated. Nevertheless, if a reference measurement is taken with a cuvette full of solvent, it can be shown that, more ore less by accident, the Beer-Lambert law works to a good approximation (Article Employing Theories Far beyond Their Limits-The Case of the (...
), but only if the index of refraction of the solvent is not too high and the solution is so diluted that this index of refraction is only marginally altered by that of the solute. Also, the thickness of the cuvette must not be too thin, otherwise the thickness variations are too small to avoid interference, which leads to electric field standing wave effects, which are very detrimental to the linearity assumed in the Beer-Lambert law, see e.g.Article The Electric Field Standing Wave Effect in Infrared Transmis...
). Actually, absorption can even decrease with increasing thickness if such effects come into play.The more the actual optical model deviates from a simple propagation of the light wave in a homogeneous medium, the larger are the deviations from the Beer-Lambert law in general. In particular, assuming a medium where scattering occurs, the breakdown of linearity can be expected and is not surprising. Unfortunately, this is a situation which cannot be solved and investigated analytically.
Of course, on top of this, chemical changes of the solution with increasing concentration like, e.g., association of molecules as well as instrumental limitations like sensitivity of the detector (Absorbance = 3 means that only 0.1 % of the light reaches the detector, reflection effects not included) and stray light etc can cause further deviations from the Beer-Lambert law...
P.S.: "Shadowing" is a concept that makes no sense in a homogeneous medium...
to dr Mayerhofer
Notice also that in many questions in Spectroscopy the question is completely vague like in this one
it only mentions " higher and low concentration of a particular metallic solution":
There is no way this can be answered as asked
I admit that the nature of the question forbids an answer. Nevertheless, some answers are also revealing... probably it would make sense to start a new "discussion" about this topic. On the other hand, it is hard these days to get from researchgate questions and discussions that match one's interests... so it makes sense to stimulate those discussions in questions who have found their followers in times when the match between interest and questions was better... or do you feel you still get the right questions that match your expertise?
Ok, here is an additional reason for deviations of Beer's (empiric) law, based on the fact that the law derived from electromagnetic theory is slightly different... ;-)
Article Beer's Law – Why Absorbance Depends (Almost) Linearly on Concentration
Simple reason is state of matter. For any matter in any state the physio-chemical properties behave linearly within certain range of reference. For example foe a matter "X" the state of substance and its physio-chemical can be varied only within a certain limit which will be specific to that substance. In this case it will follow Beers-Lambert law. If we add a specific component to the matter "X" outside that limit the physio-chemical properties of the matter changes. In case the matter itself will change its form. When matter itself changes then the spectral science will also change which will be different from the primary form matter.
Basavaraj Amogi This is an oversimplification of the situation. As I wrote above, the relation between absorbance and concentration is intrinsically non-linear. This is immediately obvious for excitations with high oscillator strength. There is, however, a way to remove this problem by using integrated absorbance which is proportional to oscillator strength (and, thereby, to concentration):
Article Beer’s law – why integrated absorbance depends linearly on c...
Istrumental deviations may result in absorbances that are smaller than theoretical. Radiation exiting from a monochromator is contaminated with small amounts of scattered or stray radiation, as a result of scattering and reflections from internal surfaces. When measurements are made in the presence of stray radiation, or with polycromatic radiation (diffferent molar absorptivities), negative deviations from Beer's Law occur, specially for higher concentrations.
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