DE19847370A1 - Use of perylene tetracarbobisimide dyes, tetraesters and tetracarboxylates are used as standard substances in fluorimetry and of bisanhydride as starting material - Google Patents

Abstract

The claims cover the use of perylene dyes and its deriveratives (perylene-3,4:9,10-tetracarbobisimides) (I), perylenetetracarboxylic tetraesters (II) and tetracarboxylates (III) are used as standard substances for fluorimetry; and perylene-3,4:9,10-tetracarboxylic bisanhydride (IV) as starting material for (III) as an analytical function for correction of the fluorimeter. The use of perylene dyes (perylene-3,4:9,10-tetracarbobisimides) (I) of formula (A; X = NR<1>; Y = NR<2> ) as standard substances for fluorimetry is claimed; R<1>, R<2> = hydrogen, (cyclo)aliphatic groups with up to 36 carbon (C) atoms or (hetero)aromatic groups, preferably with 1-4 alkyl substituents, each with up to 18 C atoms . Independent claims are also included for (a) the use of perylenetetracarboxylic tetraesters (II) as standard substances for fluorimetry; (b) the use of perylene-3,4,9,10-tetracarboxylates (III) as fluorescence standard in the aqueous phase; (c) the use of perylene-3,4:9,10-tetracarboxylic bisanhydride (IV) of formula (A; X, Y = O ) as starting material for the production of (III) by hydrolysis; (d) the use of an analytical function, preferably exponential functions, as correction for the fluorimeter.

Description

The determination of fluorescence quantum yields of dyes is by no means a routine procedure ^[1] . The measurement of absolute quantum yields requires special equipment. In addition, the measurement of the quantum yields via the fluorescence intensity, via the thermal blooming method or via the use of ultrasound methods requires considerable experience of the experimenter in order to rule out systematic measurement errors which, for. B are caused by loss of light due to reflections at the cuvette windows. An alternative to this is to calculate the fluorescence quantum yields from the easier to determine fluorescence decay times. However, this requires the precise molar extinction coefficient of the dye, which requires an analytically pure sample of the substance. This can be problematic if only very small amounts of dye are available or if the dye cannot be obtained in a stoichiometric composition.

In contrast, the determination of fluorescence quantum yields relative to a fluorescence standard can in principle even be carried out with routine fluorescence spectrometers, and many systematic errors are eliminated in this method. However, these spectrometers are usually not spectrally corrected for either the monochromator transmission or the photomultiplier sensitivity (detector sensitivity). In addition, there are no simple fluorescence standards with which the calibration can be routinely checked ("quality control"). In addition, z. For example, special care must be taken when using quinine sulfate, which is commonly used today, since the measurement conditions must be strictly observed, and its fluorescence quantum yield is not 100%, but only 54.6% ^{[2] [3] [4]} . The competing quench process is against external influences, such as. B. the measuring temperature sensitive. An uncomplicated, insensitive fluorescence standard that would fluoresce with 100% quantum yield would therefore be of interest.

Results

Perylene derivatives are generally considered as fluorescence standards, since many derivatives have extremely high fluorescence quantum yields. Of these, the perylene-3,4: 9,10-tetracarboxylic acid bisimide S-13 (1) ^{[5] is} particularly suitable because of its exceptionally high light fastness, easy solubility in organic media, low tendency to aggregate, and its high molar Extinction coefficient and its astonishingly high fluorescence quantum yield of 100%. The latter is also surprisingly not affected by air-oxygen in the solvent. The dye S-13 can easily be prepared by a one-step condensation and can be obtained with high purity by standard column chromatography - for further details see ref. ^[6] .

Chloroform is a suitable solvent for the determination of fluorescence quantum yields because many dyes in this solvent show little tendency to aggregate. The UV / Vis spectra of S-13 in chloroform are shown in Fig. 1. The fluorescence spectrum of S-13 in chloroform is structured with bands at λ _max ((I _rel ): 534.0 nm (1,000), 576.3 (0.530), 624.2 (0.115) and 681 (0.014). The position of the bands (λ _max ) can be used for a wavelength calibration of the fluorescence spectrometer and the relative intensities (I _rel ) for a spectral correction of the spectrometer sensitivity or for checking such a correction.

The UV / Vis absorption and fluorescence range of dye S-13 is a good compromise for its use as a fluorescence standard. The perylene-3,4,9,10-tetracarboxylic acid tetraester (2) is an alternative if a standard for shorter wavelengths is required: λ _max (I _rel ): 487 nm (1,000), 520 (0.739), 559 sh (0.255), 612 sh (0.047) in chloroform (see Fig. 2). The fluorescence quantum yield of 2 is also 100% and is insensitive to the usual oxygen content of the solvents. The solubility and photostability of 2 are quite sufficient for its use as a fluorescence standard, but they are not as high as that of S-13.

For even shorter wavelengths, perylene itself can be used as the fluorescence standard: λ _max (I _rel ): 442.7 nm (1,000), 472.0 (0.729), 504.4 (0.240), 542 sh (0.049) in chloroform (see Fig. 3). The fluorescence quantum yield of perylene is almost 100% (92% in ethanol ^[3] ). However, its photostability is noticeably lower than that of the other fluorescence standards presented, especially in the presence of oxygen. Perylene should therefore only be used in special cases.

Fluorescence quantum yields in the aqueous phase

When measuring fluorescence quantum yields, the same solvent should be used for the measurement sample and the reference, since part of the measurement light is lost through reflection at the cuvette windows and these light losses depend on the refractive index of the solvent. When using one and the same solvent, these losses are compensated for and need not be calculated. Accordingly, a water-soluble fluorescence standard is required for water-soluble fluorescent dyes which are important for biological systems. The tetraanion of perylene-3,4,9,10-tetracarboxylic acid (4) is suitable for this, which also fluoresces with 100% quantum yield. An aggregation of 4 in the aqueous phase is prevented by the electrostatic repulsion of the highly charged molecule. The UV / Vis spectra of 4 are similar to the spectra of tetraester 2. The photostability of 4 is high and meets the requirements for a fluorescence standard: λ _max (I _rel ): 478.9 nm (1,000), 510.6 (0.778), 547 sh ( 0.295), 591 sh (0.072) in water (see Fig. 4). However, it is already considerably smaller than that of 1. It is not necessary for the measurements to isolate the salt of 4, but the anhydride 3 can be used, which is accessible as a technical pigment (CI 71127) in pure form. 3 can be converted into 4 by the action of aqueous potassium carbonate solution.

Instructions for practical measurements

The fluorescence quantum yields of the majority of the fluorescent dyes are almost regardless of the excitation wavelength - z. B. on a large scale Quantum counters. The excitation wavelength of the majority of the Fluorescent dyes can therefore basically cover the entire range of Absorption spectrum can be chosen freely. However, the exact absorption of the Dye at the excitation wavelength for the calculation of the fluorescence quantum yields needed. However, Lambert-Beer law is best fulfilled where the slope of the Spectrum is zero (horizontal tangent) - this is due to the limited resolution of the fluorescence excitation monochromator. The fluorescence excitation should therefore preferred in the vicinity of maxima and minima of the absorption spectra of the  Fluorescent dye and the reference dye. This problem does not apply of course, if a monochromatic laser is used for excitation.

The aggregation of dyes is of particular importance for fluorescence quench processes. Chloroform as a solvent reduces the tendency for aromatics to aggregate and can be recommended as a standard solvent for fluorescence measurements, since the majority of the dyes contain aromatics as partial structures. An aggregation can be recognized by measuring the fluorescence quantum yields at two or more different dye concentrations. Another typical indication for aggregations is a change in the line shape of the dye absorption spectra, which is a Gaussian function for molecularly disperse dyes, but a Lorentz function for aggregates ^[7] . However, it must be taken into account here that, especially in dynamic exchange processes, even relatively small proportions of aggregates can significantly reduce the fluorescence quantum yield.

Oxygen dissolves in the solvents used and can be used for fluorescence quenching to lead. The fluorescence quantum yield of the dissolved perylene dyes is insensitive against air-oxygen at normal pressure - this makes working with these substances easier significantly as fluorescence standards. However, this does not apply to many other dyes, so that then degassed solutions must be used.

The absorbance (E) of the sample and the reference must be precise at the excitation wavelength be determined. For this it is recommended to use dye solutions with E about 0.3 / 1 cm Produce layer thickness at the excitation wavelength and then the E value precisely measure up. These solutions must then be diluted 1:10 for the fluorescence measurements. The use of a cuvette with a layer thickness of 10 cm would be used for the absorption measurements an alternative, but is more difficult to handle and requires more spectroscopically pure Solvent.

The reabsorption of the fluorescent light by the dye itself can be systematic Be a source of error for the measurement of fluorescence quantum yields. Highly fluorescent Dyes such as B. S-13, can be measured at low optical densities (e.g. E = 0.03 / 1 cm). However, the reabsorption must be carried out with weakly fluorescent dyes  are taken into account where higher optical densities are required for the measurements are.

The calculation of fluorescence quantum yields

Φ = (Φ _ref .fE _ref ) / (f _ref .E) (1)

If the solvent for the fluorescence sample and the reference is one and the same, then the fluorescence quantum yield of the reference can be calculated using formula (1), in which Φ is the fluorescence quantum yield of the sample and Φ _ref that of the reference. E and E _ref are the absorbances of the sample or reference at the excitation wavelength. The excitation of the sample and the reference should preferably take place at the same wavelength, so that no spectral calibration of the excitation light is required, which in turn would be faulty. f and f _ref are the integrals of the fluorescence spectra of the sample and the reference, respectively. The integration of the spectra has the further advantage that the individual intensities of the fluorescence spectra are averaged, so that the noise of the spectrometer becomes less important.

Results and discussion

The very good usability of dyes 1, 2 and 4 as fluorescence standards is through many measurements in our laboratories are documented. The substances are particularly suitable as Standards for routine fluorescence measurements. The reproducibility of the determination of Fluorescence quantum yields are 1% and better. The absolute error is 5 to 10% estimated. The relative measurement of fluorescence quantum yields presented requires very little substance, not more than is necessary for an absorption measurement, so that even of fluorescent traces the fluorescence quantum yield can be determined. The molar The extinction coefficient of the dye need not be known, it just has to the exact absorbance of the solution can be measured at the excitation wavelength.

Other derivatives of 1 and 2 as fluorescence standards

The chromophore of 1 has orbital nodes on the nitrogen atoms in HOMO and LUMO and nodes ^[8] and at least very small orbital coefficients or nodes in the neighboring orbitals (HOMO-1 and LUMO + 1). There is therefore a "closed chromophore" with respect to substituents on these atoms. This is of particular advantage for use as a fluorescence standard, since the choice of substituents on these atoms is thus free within wide limits. You can easily control the other properties of the dye with the substituents. The 1-hexylheptyl residue in 1 (S-13, "dovetail substituent" ^[8] ) z. B. easy solubility in organic solvents. The substituent is a good compromise for the practical use of the dye, since the shorter-chain homologs (e.g. the 1-pentylhexyl derivative) are less readily soluble, while the higher homologues crystallize more poorly, so that their purification is more complicated. For extreme solubility requirements, the derivative with the 1-nonyldecyl residue is still recommended, so that one then has to accept the more complex cleaning. Alternatives would be n-alkyl derivatives or cycloalkyl derivatives, the latter in particular in the area of the medium to large rings. The solubilities of these dyes are generally lower than that of 1, they are generally more difficult to prepare in high purity and have no advantages over 1. As a further alternative to 1, dyes with different chain lengths of the "dovetail residues" could be imagined. The dye is usually obtained in the synthesis as a mixture of diastereomers. This also complicates the processing or the analysis of the substances, so that this substitution also has no advantages over 1.

Fluoresce perylene bisimides with aromatic substituents on the nitrogen atoms also very strong. The phenyl derivative is extremely poorly soluble, so that it as Fluorescence standard is not very suitable. Solubility can, however, be caused by alkyl substituents be significantly increased. Examples are 2,5-di-tert-butylphenyl-, 3,5-di-tert-butylphenyl-, 2,5-di-iso-propylphenyl-, 3,5-di-iso-propylphenyl-, 2,6-di-iso-propylphenyl-, 2,4,6-tri isopropylphenyl, 2-tert-butylphenyl, 3-tert-butylphenyl, 4-tert-butylphenyl, 2- Methylphenyl, 3-methylphenyl, 4-methylphenyl, 2-methylphenyl, 2,6-dimethylphenyl, 2,5-dimethylphenyl and the 2,4,6-trimethylphenyl derivative. Combinations of these too Alkyl substituents are conceivable. Examples include 4-tert-butyl-2,6- dimethylphenyl and the 4,6-di-tert-butyl-2-methylphenyl derivative called. Higher Homologs of benzene, such as. B. the naphthyl radical or anthryl radical can be used  - to increase the solubility, the substitution with the alkyl groups mentioned is too recommend. Heterocyclic substituents are also conceivable. Examples are 2-pyridyl, 3- Pyridyl, 4-pyridyl, 2-quinolyl, 3-quinolyl, 4-quinolyl, 5-quinolyl, 6-quinolyl, 7- Quinolyl, 8-quinolyl, 1-isoquinolyl, 3-isoquinolyl, 4-isoquinolyl, 5-isoquinolyl, 6- Isoquinolyl, 7-isoquinolyl, 8-isoquinolyl, 2-thiophenyl and 3-thiophenyl radical. Also at these heterocyclically substituted dyes is advantageously an increase in Solubility achieved with the alkyl groups mentioned under the phenyl radical. The aromatic Substituted dyes generally have. no advantages over dye 1 because its Photostabilities are consistently lower than that of 1 and because of it Fluorescence quantum yields are consistently slightly lower than that of 1.

The alternative substituents mentioned under 1 can also be used as an alcohol component in Dyes are used which are analogous to 2 esters. But these are significant more difficult to synthesize than 2. In addition, aromatic esters are generally. more unstable against Hydrolysis or saponification as purely aliphatic esters. It is therefore the dye 2 of the Preferred.

Experimental part Spectral calibration of fluorescence spectrometers

We used the Perkin Elmer 3000 fluorescence spectrometer for all measurements. The spectral sensitivity of the photomultiplier detector can be done using the S-13 dye. Calibrated spectra of S-13 were obtained with a SPEX Fluorolog spectrometer using a calibration lamp from the Swedish Institute for Standards. Our spectrometer could be corrected point by point with a correction file. However, this would lower the signal-to-noise ratio of the spectra, since the correction table itself also contains noise and other deviations. Better results were achieved by using an analytical correction function.

I _corr = I.exp ((λ - 540) /47.7) (λ <540 nm) (2)

We found that equation (2) represents a useful global spectral correction of the built-in photomultiplier (9781R) and monochromator transmission for the wavelength range greater than 540 nm. Here I _{corr is} the corrected fluorescence intensity and I is the uncorrected intensity at the wavelength (λ) in nm. The fluorescence spectrum of S-13 can be e.g. B. are hereby corrected sufficiently. However, the photomultiplier sensitivity drops considerably at wavelengths greater than 650 nm, so that it becomes difficult to obtain correct spectra in the long-wave spectral range.

I _ph. = I.1.25. (1 + exp ((λ - 403.04) / - 370). (1 + exp ((λ - 413.04) / 130). (1 + exp ((λ - 842) / 12) (3)

I _corr = I _ph. .exp (0.003. (λ - 540)) (λ <540 nm) (4)

Better results are obtained if the detector is replaced by a red-sensitive photomultiplier (R928, Hamamatsu), with which the entire spectral range of the spectrometer is accessible. However, the spectral correction is then complicated by the more complex characteristic of the photomultiplier: first we corrected the spectral sensitivity of the detector itself with equation (3), in which I _ph. the corrected photomultiplier sensitivity is: For wavelengths greater than 540 nm, the decreasing monochromator transmission must also be taken into account, so that the additional correction by equation (4) is necessary. Together, this results in a good spectral correction for the entire range of the spectrometer from 250 to 800 nm. It can be expected that similar correction functions can also be used for other spectrometers.

An analogous correction could also be made for the fluorescence excitation spectrum. However, the calibration curve takes on due to the spectral characteristics of the excitation lamp complex course. A correction table is therefore preferable for this - but it is only of importance for fluorescence excitation spectra, and not for fluorescence spectra and the measurement of fluorescence quantum yields, because then at a constant Fluorescence excitation wavelength is measured.  

Fluorescence standards

The dyes S-13 (1 ^[9] , RN 110590-84-6) and 2 ^[10] were prepared according to the literature and purified by column chromatography (silica gel / chloroform). At 1, a yellow leader should be carefully removed because it contains an efficient fluorescence quencher. The quench effect is not so important for solutions, but more pronounced for dyes in the solid phase.

The dye 4 was prepared by an alkaline hydrolysis of the easily accessible anhydride 3 [RN 128-69-8, CI 71127]. A 1/10 NK ₂ CO ₃ solution was prepared for this by making 1,382 g of anhydrous potassium carbonate to 100 ml with double-distilled water. A micro spatula tip 3 (a few milligrams) was suspended in 10 ml of the 1/10 NK ₂ CO ₃ solution and left to stand until it turned yellow with green fluorescence. The mixture was filtered through a plugged cotton filter or through a D 5 glass frit to remove the excess of 3, discarding the first milliliters of the filtrate. The extinction of this solution was brought to about 0.3 (437 nm, 1 cm layer thickness) by dilution with the 1/10 NK ₃ CO ₃ solution and measured exactly. 1.00 ml of this solution was made up to 10.0 ml with the 1/10 NK ₂ CO ₃ solution and used for the fluorescence measurements of 4.

For the fluorescence quantum yields of 1, 2 and 4, 100% were surprisingly found (c = 6.10 ^-7 mol.1 ^-1 in chloroform for 1, 7.10 ^-7 mol.1 ^-1 for 2 and 7.10 ^-7 mol.1 ^-1 for 4 in water / 0.1 MK ₂ CO ₃ ) found with an accuracy of 1% against quinine sulfate. The photochemical stability of 1 and 2 is so high that stock solutions can be kept for a long period of time. This is astonishing for fluorescent dyes, since fading is usually expected to be considerably faster, so that the solutions are more difficult to handle. The photostability of 4 is significantly lower than that of the other standards, but still so high that stock solutions can be used without any problems, but which should be kept in the dark for long periods. The fluorescence of the three dyes is not quenched by atmospheric oxygen, so that the solutions can be handled simply and easily in the air.

The structured spectra of 1, 2 and 4 make it possible to excite fluorescence at a variety of wavelengths. However, in order to achieve maximum accuracy, it is recommended to select excitation wavelengths in the vicinity of absorption maxima or minima, since maximum accuracy of the measurement is then achieved with a limited resolution of the excitation monochromator. This is due to the fact that Lambert-Beer law applies strictly only to monochromatic light radiation. The following wavelengths are recommended for the excitation: 1: 490.0 (526.6 _max 504.6 _min , 469.0 _min and 458.6 _max in chloroform), 2: 442.4 (471.1 _max , 454.7 _min and 420 _sh. In chloroform) and 4: 437.4 (465.7 _max , 449.2 _min and 415 _sh. In water). The concentration of the dye solutions is not critical, but there is an aggregation with fluorescence quenching at very high concentrations. An absorbance of 0.03 (1 cm layer thickness) is recommended for the excitation wavelength to measure the fluorescence quantum yields.

The higher homologues ^[11] of 1 with aliphatic side chains have the same fluorescence properties, but are somewhat more difficult to display or clean than 1. They can be used when solubilities higher than 1 are required.

literature

1. See for example: OS Wolfbeis, Fluorescence Spectroscopy. New Methods and Applications, Springer Verlag, Berlin 1993; ISBN 3-540-SS281-2.
2. WH Melhuish, J. Chem. Phys. 1960, 64, 762.
3. G. Weber, FWJ Teale, Trans. Faraday. Soc. 1958, S4, 640.
4. DF Eaton, Pure Appl. Chem. 1988, 7, 1107.
5. H. Langhals Heterocycles 1995, 40, 477.
6. H. Langhals, J. Karolin, LB-Å. Johansson, J. Chem. Soc., Faraday Trans., In press.
7. EW Knapp, Chem. Phys. 1984, 85, 73.
8. H. Langhals, S. Demmig, H. Huber, Spectrochim. Acta 1988, 44A, 1189.
9. S. Demmig, H. Langhals, Chem. Ber 1988, 121, 225.
10. BASF (Erf. H. Eilingsfeld, M. Potsch) DOS 25 12516 (Sept. 30, 1976); Chem. Abstr. 1977, 86, P31008p (see also L. Feiler, H. Langhals, K. Polborn, Liebigs Ann. 1995, 1229).
11. H. Langhals, S. Demmig, T. Potrawa, J. Prakt. Chem. 1991, 333, 733.

formula 1

Reference list

Fig.

1. UV / Vis absorption and fluorescence spectra of 1 in chloroform (color coordinates for the absorption: x = 0.3616, y = 0.3139, Y = 81.24; 2 °, standard light C, T _min

= 0.1).

Fig.

2. UV / Vis absorption and fluorescence spectra of 2 in chloroform (color coordinates for absorption: x = 0.3884, y = 0.4504, Y = 94.41; 2 °, standard light C, T _min

= 0.1).

Fig.

3. UV / Vis absorption and fluorescence spectra of perylene in chloroform (color coordinates for the absorption: x = 0.3320, y = 0.3627, Y = 99.45; 2 °, standard light C, T _min

= 0.1).

Fig.

4. UV / Vis absorption and fluorescence spectra of 4 in 0.1 N aqueous K ₂

CO ₃

- Solution (color coordinates for absorption: x = 0.3820, y = 0.4443, Y = 95.17; 2 °, standard light C, T _min

= 0.1).

Claims (5)

1. Use of perylene dyes (perylene-3,4: 9,10-tetracarboxylic acid bisimides) of the general formula 5 as calibration substances for fluorimetry, in which the radicals R ¹ and R ^{2 can be the} same or different. The radicals R ¹ and R ² can be hydrogen, aliphatic, also branched or cyclic, with one to 36 C atoms, or they can be aromatic, then in turn preferably with one to four alkyl substituents, each with one to 18 C Atoms, which can be linear, branched or cyclic, or they can be heteroaromatic, then preferably with the radicals mentioned for the aromatic substituents.
Examples of aliphatic radicals R ¹ and R ² in 5 are methyl, ethyl, 1-propyl, 1-butyl, 1-pentyl, 1-hexyl, 1-heptyl, 1-octyl, 1-nonyl, 1-decyl, 1-undecyl, 1-dodecyl, 1-tridecyl, 1-tetradecyl, 1-pentadecyl, 1-hexadecyl, 1-heptadecyl, 1-octadecyl, 1-nonadecyl, 1-eicosyl, 1- Pentatriacontyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, cyclododecyl, cyclotridecyl, cyclotetradecyl, cyclopentadecyl, cyclohexadecyl, cyclohexadecyl, cyclohexadecyl, cyclohexadecyl, cyclohexadecyl, cyclohexadecyl, cyclohexadecyl, cyclohexadecyl, cyclohexadecyl, cyclohexadecyl, cyclohexadecyl, cyclohexadecyl, cyclohexadecyl, cyclohexadecyl, cyclohexadecyl, cyclohexadecyl, cyclohexadecyl, cyclohexadecyl, cyclohexadecyl, cyclohexadecyl, cyclohexadecyl, cyclohexadecyl, cyclohexadecyl and cyclohexadecyl Cyclononadecyl, cycloeicosyl, 3-ethylpropyl, 3-ethylbutyl, 3-ethylpentyl, 3-ethylhexyl, 3-ethylheptyl, 3-ethyloctyl, 3-ethylnonyl, 3-ethyldecyl, iso-propyl, 1-ethylpropyl, 1-propylbutyl, 1-butylpentyl, 1-pentylhexyl, 1-hexylheptyl, 1-heptyloctyl, 1-octylnonyl, 1-nonyldecyl, 1-decylundecyl, 1-undecyldodecyl, 1- dodecyltridecyl, 1-heptadecyloctadecyl and the 1-octadecylnonadecyl radical. R ¹ = R ² = 1-hexylheptyl (1) and 1-nonyldecyl is preferred. Of these, R ¹ = R ² = 1-hexylheptyl (1) is most preferred.
Examples of aromatic radicals R ¹ and R ² in 5 are 2,5-di-tert-butylphenyl-, 3,5-di-tert-butylphenyl-, 2,5-di-iso-propylphenyl-, 3,5- Di-iso-propylphenyl-, 2,6-di-iso-propylphenyl-, 2,4,6-tri-iso-propylphenyl-, 2-tert-butylphenyl-, 3-tert-butylphenyl-, 4-tert-butylphenyl -, 2-methylphenyl, 3-methylphenyl, 4-methylphenyl, 2,6-dimethylphenyl, 2,5-dimethylphenyl and the 2,4,6-trimethylphenyl radical. Combinations of these alkyl substituents are also conceivable - examples include the 4-tert-butyl-2,6-dimethylphenyl and the 4,6-di-tert-butyl-2-methylphenyl derivative. Higher homologs of benzene, such as. B. the naphthyl radical or anthryl radical can be used - the substitution with the alkyl groups mentioned is recommended to increase solubility.
Examples of heteroaromatic radicals R ¹ and R ² in 5 are the 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-quinolyl, 3-quinolyl, 4-quinolyl, 5-quinolyl, 6-quinolyl -, 7-quinolyl, 8-quinolyl, 1-isoquinolyl, 3-isoquinolyl, 4-isoquinolyl, 5-isoquinolyl, 6-isoquinolyl, 7-isoquinolyl, 8-isoquinolyl, 2-thiophenyl - and 3-thiophenyl radical. With these heterocyclically substituted dyes, too, the solubility is expediently increased with the alkyl groups mentioned under the phenyl radical. 2. Use of perylene tetracarboxylic acid tetraesters of the general formula 6 as calibration substances for fluorimetry, in which the radicals R ¹ to R ^{4 can be the} same or different. The radicals R ¹ to R ⁴ can be hydrogen, aliphatic, also branched or cyclic, with one to 36 C atoms, or they can be aromatic, then in turn preferably with one to four alkyl substituents, each with one to 18 C Atoms, which can be linear, branched or cyclic, or they can be heteroaromatic, then preferably with the radicals mentioned for the aromatic substituents.
Examples of substituents are the radicals mentioned under 1. R ¹ to R ⁴ are preferably methyl. 3. Use of the perylene-3,4,9,10-tetracarboxylate (4), preferably as the potassium, sodium or ammonium salt of a tertiary amine, as a fluorescence standard in the aqueous phase.
4. Use of the perylene-3,4: 9,10-tetracarboxylic acid bisanhydride (3) as a starting material for the preparation of the fluorescence standard 4 by hydrolysis.
Preferred hydrolysis media are aqueous potassium carbonate solution, aqueous sodium carbonate solution, aqueous KOH solution and aqueous NaOH solution; most preferred is aqueous potassium carbonate solution. Preferred concentrations of the hydrolysis medium are 1/100 to 1 molar, most preferred is 1/10 molar. 5. Use of an analytical function as a correction for the fluorimeter. Are preferred Exponential functions.