Dye-Aggregation
The absorption of light causes dye molecules to enter an electronically excited state. The absorbed energy is only stored for a short time and can be radiated again after the lifetime of the excited state, e.g. as fluorescence.
In a dye solution, the excited dye molecules, which can be regarded as point dipoles or oscillators, do not influence each other if the distances between them are large enough.
At a mean distance of about 5 - 10 nm between the chromophores, the oscillators' "radiation field" only has an influence. This type of interaction between two dye molecules can be described by the model of Förster‘s resonance energy transfer (FRET).
If the distance between the chromophores becomes even smaller, e.g. in a very concentrated solution, the individual oscillators can strongly influence each other by electrostatic interaction. Due to the intermolecular interaction of the individual dye molecules, both the absorption and fluorescence behavior of such a dye solution change strongly.
Rhodamine 6G in water
The UV/Vis spectrum of a concentrated aqueous solution of rhodamine 6G shows a shoulder at the short-wave flank of the main absorption band. Changing the concentration (c) by diluting the solution and increasing the optical pathlenght (d) of the cuvette to make up for the dilution, i.e. the same absorbance would always be expected according to the Lambert-Beer law, the following spectra can be observed:
The occurrence of an isosbestic point - the change in concentration of all substances involved is linear, dE/dc = 0 - shows that two (or more) species are in a dynamic equilibrium with each other.
The dissociation or association/complexation constant can be determined experimentally: In a dilution series, in which the dilution of the solution is always compensated by the change of the pathlenght, an "effective extinction coefficient" can be calculated via the measured absorbance at the (monomer) maximum with knowledge of the dilution factor and the initial sample concentration. This sample concentration is determined from the UV spectrum of a very diluted solution where no dimerization takes place. Since the absorptions of different species in the Lambert-Beer law behave additively, an effective extinction or an effective extinction coefficient can be written down with knowledge of the underlying reaction. By parametric variation of the dissociation constant and a graphic analysis, a straight line is obtained from whose slope and axis section the extinction coefficients of monomer and dimer can be determined.
Hydrophobic interaction
The aggregation of organic dyes occurs particularly in water or solvents with high ionic strength. The main reason are intermolecular van der Waals forces, the so called "hydrophobic interaction". The lipophilic dye molecules try to avoid the hydrophilic water molecules by showing the hydrate shell the smallest possible surface area. This phenomenon is also responsible for the adsorption of dyes on glass surfaces or non-specific bonding to substrate molecules.
The tendency to form dimers or higher aggregates is dependent on
- any electrolytes (salts) present, especially when ion pairs (dye cation and counter ions) are formed in organic solvents such as chloroform
- temperature - at higher temperatures the thermal movement makes aggregation more difficult
- the molecular structure of the dye - dyes with hydrophilic groups, e.g. ATTO 488, ATTO 532, ATTO 542 etc., show no aggregation in contrast to more hydrophobic dyes such as ATTO Rho6G, ATTO Rho11, ATTO Rho12 etc. in aqueous solution:
Since this is a dynamic equilibrium, the dimers can be reconverted into monomers by diluting the solution. The "monomer spectrum" is reached when the measured absorption spectrum does not change with further dilution and the corresponding increase of the optical pathway. For most hydrophobic ATTO dyes, this is the case with an absorbance of approx. 0.04 (layer thickness 1 cm; c = 10-7 - 10-6 mol/l).
Intramolecular interaction in protein conjugates / DOL determination
The reaction of dye NHS ester with the amino groups of a protein may produce dye conjugates in which the covalently bound dye molecules are closely adjacent and can interact with each other. This can lead to a strong change in the absorption spectrum, as can be easily seen in the example of the ATTO 565 steptavidin conjugate:
In the conjugate spectrum, an additional short-wave absorption band is observed analogous to the "dimer band" of an aqueous dye solution with a sufficiently high concentration. Since this is an intramolecular interaction of the covalently bound dye molecules, the absorption spectrum does not change when the conjugate solution is diluted!
The determination of the degree of labeling (DOL) for such cases is described in our user guide & protocols "Procedures".
There are two main types of aggregates:
H aggregates (H = hypsochrome)
This type of aggregation occurs when two or more dye molecules are positioned with their transition dipole moments (usually along the longitudinal axis of the chromophoric system in the S0-S1 transition) oriented parallel to each other. In contrast to monomer absorption, a hypsochromically shifted absorption band is observed.
Due to the spatial proximity, the electronic orbitals influence each other and the two molecules have to be considered together as one unit. The energy levels are split up and the absorption allowed by quantum mechanical rules is of higher energy and therefore occurs at shorter wavelengths. From this energetically higher lying excitation state a fast internal conversion (IC) takes place, so that fluorescence is no longer possible.
J aggregates (according to E.E. Jelley)
This type of aggregation results in a long-wave shift of the absorption band, which is associated with a significant reduction in the half-width of the band.
J aggregates are often found in polymethine dyes such as cyanines, merocyanines or similar chromophores. Jelley and Scheibe observed the phenomenon for the first time independently of each other on the dye pseudoisocyanine.
Different types were proposed for the model-like description of the "supramolecular polymer" formed by the combination of the individual dyes. The simplest description of the molecular relationships is the idea that the individual molecules arrange one behind the other, so that the transition dipole moments are also in-line. The collective consideration of the molecules results in a splitting of the energy levels: The quantum-mechanically allowed transition is now energetically lower, which explains the long-wave shift of the absorption band.
The aggregation can be strongly influenced by the solvent composition, the addition of salts or additives and the concentration of the dye. Under ideal conditions, the extremely narrow absorption band can be observed in the UV/Vis spectrum. In contrast to H aggregates, fluorescence is possible with this type of aggregation, especially at lower temperatures: However, the maximum of the very narrow emission band is only a few nanometers red-shifted with respect to the absorption maximum.
Depending on the experimental conditions, a "broadening" of the absorption band is reported in the literature, which is explained, among other things, by the inclusion of the forbidden electron transfer of the J aggregate.
ATTO 488 marked phospholipids
Solutions of ATTO 488 labeled phospholipids in chloroform at first surprise with their unexpected color: Instead of light yellow with bright green fluorescence, the blunt solution appears pink to magenta. The long-wave shifted absorption can be explained by the presence of J aggregates. When the solution in question is diluted with methanol, the color changes to yellow and becomes strongly fluorescent. By changing the solvent composition, the aggregates are reduced.
The following figures show solutions of ATTO 488 marked 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamines (DPPE) in pure chloroform and in a solvent mixture of chloroform/methanol (8:2, V/V):
On the left, both solutions are shown in normal daylight. On the right, the green fluorescence of the solution mixed with methanol is particularly well visible when irradiated with UV light (366 nm).
Literatur:
E. Jelley, Spectral Absorption and Fluorescence of Dyes in the Molecular State, Nature 138, 1009 (1936).
G. Scheibe, Über die Veränderlichkeit der Absorptionsspektren in Lösungen und die Nebenvalenzen als ihre Ursache, Angewandte Chemie 50, 212 (1937).
T. Förster, Energiewanderung und Fluoreszenz, Die Naturwissenschaften 33, 166 (1946).
M. Kasha, H.R. Rawls, M. Ashraf El-Bayoumi, The Exciton Model in Molecular Spectroscopy, Pure Appl. Chem. 11, 371 (1965).
O. Valdes-Aguilera, D.C. Neckers, Aggregation Phenomena in Xanthene Dyes, Acc. Chem. Res. 22, 171 (1989).
J. Hernando et al., Excitonic Behavior of Rhodamine Dimers: A Single-Molecule Study, J. Phys. Chem. A 107, 43 (2003).
V.I. Gavrilenko, M.A. Noginov, Ab initio study of optical properties of rhodamine 6G molecular dimers, J. Chem. Phys. 124, 0044301 (2006).
In a dye solution, the excited dye molecules, which can be regarded as point dipoles or oscillators, do not influence each other if the distances between them are large enough.
At a mean distance of about 5 - 10 nm between the chromophores, the oscillators' "radiation field" only has an influence. This type of interaction between two dye molecules can be described by the model of Förster‘s resonance energy transfer (FRET).
If the distance between the chromophores becomes even smaller, e.g. in a very concentrated solution, the individual oscillators can strongly influence each other by electrostatic interaction. Due to the intermolecular interaction of the individual dye molecules, both the absorption and fluorescence behavior of such a dye solution change strongly.
Rhodamine 6G in water
The UV/Vis spectrum of a concentrated aqueous solution of rhodamine 6G shows a shoulder at the short-wave flank of the main absorption band. Changing the concentration (c) by diluting the solution and increasing the optical pathlenght (d) of the cuvette to make up for the dilution, i.e. the same absorbance would always be expected according to the Lambert-Beer law, the following spectra can be observed:
The occurrence of an isosbestic point - the change in concentration of all substances involved is linear, dE/dc = 0 - shows that two (or more) species are in a dynamic equilibrium with each other.
The dissociation or association/complexation constant can be determined experimentally: In a dilution series, in which the dilution of the solution is always compensated by the change of the pathlenght, an "effective extinction coefficient" can be calculated via the measured absorbance at the (monomer) maximum with knowledge of the dilution factor and the initial sample concentration. This sample concentration is determined from the UV spectrum of a very diluted solution where no dimerization takes place. Since the absorptions of different species in the Lambert-Beer law behave additively, an effective extinction or an effective extinction coefficient can be written down with knowledge of the underlying reaction. By parametric variation of the dissociation constant and a graphic analysis, a straight line is obtained from whose slope and axis section the extinction coefficients of monomer and dimer can be determined.
Hydrophobic interaction
The aggregation of organic dyes occurs particularly in water or solvents with high ionic strength. The main reason are intermolecular van der Waals forces, the so called "hydrophobic interaction". The lipophilic dye molecules try to avoid the hydrophilic water molecules by showing the hydrate shell the smallest possible surface area. This phenomenon is also responsible for the adsorption of dyes on glass surfaces or non-specific bonding to substrate molecules.
The tendency to form dimers or higher aggregates is dependent on
- the concentration of the dye - the higher the concentration, the greater the aggregation
- the solvent - in water or methanol, unlike ethanol or other organic solvents, aggregation can be observed more frequently. This is impressively demonstrated by the comparison of the absorption spectra of equal concentrated solutions of ATTO 565 in aqueous PBS buffer (pH 7.4) and ethanol with trifluoroacetic acid (TFAc):
- the solvent - in water or methanol, unlike ethanol or other organic solvents, aggregation can be observed more frequently. This is impressively demonstrated by the comparison of the absorption spectra of equal concentrated solutions of ATTO 565 in aqueous PBS buffer (pH 7.4) and ethanol with trifluoroacetic acid (TFAc):
- any electrolytes (salts) present, especially when ion pairs (dye cation and counter ions) are formed in organic solvents such as chloroform
- temperature - at higher temperatures the thermal movement makes aggregation more difficult
- the molecular structure of the dye - dyes with hydrophilic groups, e.g. ATTO 488, ATTO 532, ATTO 542 etc., show no aggregation in contrast to more hydrophobic dyes such as ATTO Rho6G, ATTO Rho11, ATTO Rho12 etc. in aqueous solution:
Since this is a dynamic equilibrium, the dimers can be reconverted into monomers by diluting the solution. The "monomer spectrum" is reached when the measured absorption spectrum does not change with further dilution and the corresponding increase of the optical pathway. For most hydrophobic ATTO dyes, this is the case with an absorbance of approx. 0.04 (layer thickness 1 cm; c = 10-7 - 10-6 mol/l).
Intramolecular interaction in protein conjugates / DOL determination
The reaction of dye NHS ester with the amino groups of a protein may produce dye conjugates in which the covalently bound dye molecules are closely adjacent and can interact with each other. This can lead to a strong change in the absorption spectrum, as can be easily seen in the example of the ATTO 565 steptavidin conjugate:
In the conjugate spectrum, an additional short-wave absorption band is observed analogous to the "dimer band" of an aqueous dye solution with a sufficiently high concentration. Since this is an intramolecular interaction of the covalently bound dye molecules, the absorption spectrum does not change when the conjugate solution is diluted!
The determination of the degree of labeling (DOL) for such cases is described in our user guide & protocols "Procedures".
There are two main types of aggregates:
H aggregates (H = hypsochrome)
This type of aggregation occurs when two or more dye molecules are positioned with their transition dipole moments (usually along the longitudinal axis of the chromophoric system in the S0-S1 transition) oriented parallel to each other. In contrast to monomer absorption, a hypsochromically shifted absorption band is observed.
Due to the spatial proximity, the electronic orbitals influence each other and the two molecules have to be considered together as one unit. The energy levels are split up and the absorption allowed by quantum mechanical rules is of higher energy and therefore occurs at shorter wavelengths. From this energetically higher lying excitation state a fast internal conversion (IC) takes place, so that fluorescence is no longer possible.
J aggregates (according to E.E. Jelley)
This type of aggregation results in a long-wave shift of the absorption band, which is associated with a significant reduction in the half-width of the band.
J aggregates are often found in polymethine dyes such as cyanines, merocyanines or similar chromophores. Jelley and Scheibe observed the phenomenon for the first time independently of each other on the dye pseudoisocyanine.
Different types were proposed for the model-like description of the "supramolecular polymer" formed by the combination of the individual dyes. The simplest description of the molecular relationships is the idea that the individual molecules arrange one behind the other, so that the transition dipole moments are also in-line. The collective consideration of the molecules results in a splitting of the energy levels: The quantum-mechanically allowed transition is now energetically lower, which explains the long-wave shift of the absorption band.
The aggregation can be strongly influenced by the solvent composition, the addition of salts or additives and the concentration of the dye. Under ideal conditions, the extremely narrow absorption band can be observed in the UV/Vis spectrum. In contrast to H aggregates, fluorescence is possible with this type of aggregation, especially at lower temperatures: However, the maximum of the very narrow emission band is only a few nanometers red-shifted with respect to the absorption maximum.
Depending on the experimental conditions, a "broadening" of the absorption band is reported in the literature, which is explained, among other things, by the inclusion of the forbidden electron transfer of the J aggregate.
ATTO 488 marked phospholipids
Solutions of ATTO 488 labeled phospholipids in chloroform at first surprise with their unexpected color: Instead of light yellow with bright green fluorescence, the blunt solution appears pink to magenta. The long-wave shifted absorption can be explained by the presence of J aggregates. When the solution in question is diluted with methanol, the color changes to yellow and becomes strongly fluorescent. By changing the solvent composition, the aggregates are reduced.
The following figures show solutions of ATTO 488 marked 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamines (DPPE) in pure chloroform and in a solvent mixture of chloroform/methanol (8:2, V/V):
On the left, both solutions are shown in normal daylight. On the right, the green fluorescence of the solution mixed with methanol is particularly well visible when irradiated with UV light (366 nm).
Literatur:
E. Jelley, Spectral Absorption and Fluorescence of Dyes in the Molecular State, Nature 138, 1009 (1936).
G. Scheibe, Über die Veränderlichkeit der Absorptionsspektren in Lösungen und die Nebenvalenzen als ihre Ursache, Angewandte Chemie 50, 212 (1937).
T. Förster, Energiewanderung und Fluoreszenz, Die Naturwissenschaften 33, 166 (1946).
M. Kasha, H.R. Rawls, M. Ashraf El-Bayoumi, The Exciton Model in Molecular Spectroscopy, Pure Appl. Chem. 11, 371 (1965).
O. Valdes-Aguilera, D.C. Neckers, Aggregation Phenomena in Xanthene Dyes, Acc. Chem. Res. 22, 171 (1989).
J. Hernando et al., Excitonic Behavior of Rhodamine Dimers: A Single-Molecule Study, J. Phys. Chem. A 107, 43 (2003).
V.I. Gavrilenko, M.A. Noginov, Ab initio study of optical properties of rhodamine 6G molecular dimers, J. Chem. Phys. 124, 0044301 (2006).