Fluorescence Efficiency of Laser Dyes*

The fluorescence efficiency of xanthene dyes, oxazine dyes, and 7-aminocoumarins is discussed. Relations with the molecular structure are pointed out and dependence on solvent and temperature is explained. Several new fluorescence standards are suggested.


Introduction
The rece nt de velopment of the dye laser [1] 1 has opened up an important new field of applications for organic dyes. This has led to a rene wed inte rest in the theory of nonradiative transitions in dyes and also to the synthesis of new highly fluores cent dyes. In this article we review briefly the relations between fluor· escence and molec ular structure in the most important classes of laser dyes: xanthenes , oxazines, and 7·amino· coumarins. Following this discussion specific sug· gestions for improved fluorescence standards are made. 2

Xanthene Dyes
The chromophore of xanthene dyes has typically the followin g structures Depending on the end groups, in particular number and type of the substituents R, the maximum of the main absorption band falls somewhere in the range 480-580 nm. The transition moment is parallel to the long axis of the molecule [2]. With R '= H and amino end groups the dyes are called pyronins. Due to a con· venient syntheses with phthalic anhydride many xanthene dyes have R I = carboxyphenyl and are called fluorescein or rhodamines ( fig. 1). The methyl substi· tuents of Rhodamine 6G have practically no influen ce on the optical properties of the dye except for the dimerization in aqueous solution which we are not concerned with here. The absorption maximum of the main band occurs almost at the same wavelength in pyronins and rhodamines, if the end groups are identical. The absorption band near 350 nm is stronger in rhodamines than in pyronins, and rhodamines show a slightly larger Stokes shift than pyronins. The chemical stability of rhodamines is ge nerally suo perior, as pyronins in alkaline solution are readily oxidized by dissolved oxygen to form a colorless, blue fluores cing xanthone. Rhodamines like Rhodamine B react to pH variations in an interesting manner. The carboxyl group is completely protonated in acidic solution, but dissociates in alkaline solution_ The negative charge has an inductive effect on the central carbon atom of the chromophore. The maxima of the main absorption band and of the fluorescence are shifted to shorter wavelengths, and the extinction coefficient at the absorption maximum is slightly reduced. While these effects are rather weak in aqueous solution [3], the wavelength shift amounts to about 10 nm in polar organic solvents (methanol, ethanol, etc.) [4,5,6,7]. When Rhodamine B, which is usually obtained as the hydrochloride, is dissolved in ethanol, the relative amounts of the two forms of the dye are determined by this acid-base equilibrium. The dissociation increases on dilution causing a shift of absorption and fluorescence to shorter wavelengths_ This concentration dependence of the spectra is therefore not due to a monomerdimer equilibrium , as was claimed in recent papers [8,9]. In less polar organic solvents (e.g_, acetone) the unprotonated (zwitterionic) dye undergoes a reversible conversion to a lactone: Because the conjugation of the chromophore is interrupted. this is a colorless compound. On addition of acid or complex-forming metal ions the chromophore is regenerated. If the carboxyl group is esterified as in Rhodamine 6G, all these reactions do not take place, and the absorption maximum is independent of pH and concentration (except for aqueous solutions, where aggregation takes place, and for strongly acidic conditions, where protonation of the amino groups occurs).
Because of the symmetry of the rhodamine chromophore, there is no dipole moment parallel to its long axis. As a consequence, the Stokes shift is small, and the fluorescence band overlaps strongly the absorption band ( fig. 2) [10]. Also, there is only little variation of absorption and fluorescence maxima with solvent polarity [4,5]. Provided there are no heavy-atom substituents, the rate of intersystem crossing from S 1 is very low in xanthene dyes, which is in accordance with our loop rule [11]. Phosphorescence is weak even in low temperttl + H ature glasses and has a lifetime in the order of 0.1 s, which is short compared with, for instance, acriflavine and some aromatic compounds.
The fluorescence of rhodamines is quenched externally by I -and SCN -, less efficiently by Brand Cl -. No quenching was observed by CI04 -and BF 4 -[4, 5]. The quenching process apparently involves a charge transfer from the anion to the excited dye molecule. It is not a heavy-atom effect. In polar solvents like ethanol, Rhodamine 6G-iodide is completely dissociated at a concentration of 10-4 molll and no quenching takes place, because the lifetime of the excited state is of the order of a few nanoseconds, much shorter than the diffusion time the quencher would need to reach an excited dye molecule. In less polar solvents like chloroform the dye salt does not dissociate and its fluorescence is totally quenched. The perchlorate of Rhodamine 6G behaves strikingly differently: its fluorescence efficiency is independent [12] of solvent polarity (0.95 based on the value 0.90 for fluorescein). It is, however, reduced by heavy-atom solvents (0.40 in iodomethane), and the fluorescence of this dye is completely quenched by nitrobenzene, presumably due to the high electron affinity of this solvent.
There are two structural features that influence the rate of internal conversion in xanthene dyes: mobility of the amino groups and hydrogen vibrations. ,- 4 00 Wavelength 6 00 700 Ahsor pti on s pec trum (E mo lar decadi c e xtin ction coe ffi c ient): (IU ant um s pectrum or flu oresce nce (a rbit ra ry unit s).
We found th a t th e fluoresce nce e fficie nc y of rhodamines tha t carry two alkyl subs titu e nts at eac h nitroge n, e.g. , Rhodamin e B, vari es stron gly with solve nt and tem pera ture [4 , 5, 11]. We ascribe thi s to mobility of th e amino groups. If th e amino groups are ri gidized as in th e ne w dye Rhod a min e 101 (A abs=577 nm in ethanol) th e quantum effi cie nc y is practically unity,

Rhodamine 101
independent of solvent and te mperature. It is inter· esting that the amino groups are not mobile when they are less than fully alkylated (Rhodamine 6C, Rhodamine llO). Howe ver, in such dyes there is a proba-dyes , it is noti ceable for in stance in Rhodamine 6C.
On soluti on in O-d e ute rated eth a nol, H is exc han ged for D in th e amino groups of th e dye. With the greater mass of de uterium th e nonradi a tiv e deca y is less ]jkely and the fluorescence e ffi cienc y increases from 0.95 to 0.99.

Oxazine Dyes
Whe n the ce ntral carbon a tom in the xanthene chromophore is re placed b y nitrogen , th e c hromophore of an oxazine dye is obtained. The ce ntral N-atom acts as a sink for the 7T-electrons, cau sin g a wavele ngth shift of about 80 nm to the red . As within the xanthe ne class the absorption of oxazines also shifts to the red with increasing alkylation of the amino groups (fi g. 3).   Some oxazine dyes have a structure modified by an added benzo group (fig. 3). This causes a slight red shift of absorption and fluorescence. Furthermore, the shape of the absorption spectrum is different than in other oxazine dyes and depends on the temperature [1 3]. These effects are probably caused by steric interference of the amino group with a hydrogen of the added benzo group [11].
The triplet yield of oxazines is generally very low in accordance with the loop rule_ No phosphorescence has been observed in these dyes. The fluorescence quenching processes, discussed for xanthene dyes, are also found with oxazines. Owing to the smaller energy difference between 51 and 50, internal conversion plays a greater role in oxazines than in rhodamines. Thus the fluorescence efficiency of Oxazine I-perchlorate is less than 0_1 in ethanol, but much higher in dichloromethane and in 1,2-dichlorobenzene_ Likewise, the effect of hydrogen vibrations is much more pronounced. The fluorescence efficiency of Oxazine 4 is a factor of 2 higher in O-deuterated ethanol than in normal ethanol.

7 -Aminocoumarins
The most important laser dyes in the blue and green region of the spectrum are coumarin derivatives that have an amino group in 7-position: The chromophore is not symmetric as in the xanthenes and oxazines discussed above. The ground state can be described by structure A, the excited state 5 I by B. While there is some dipole moment in the ground state, it is much greater in the excited state. In the excited state 51, the keto group is more basic than the amino group and thus is protonated if sufficient acid is present so that the diffusion is faster than the decay of 51. Frequently a new fluorescence band appears due to the protonated form. Most coumarins are very soluble in organic solvents, but insoluble in water. However, derivatives have been reported recently that are highly soluble in water ( fig. 6) [18].

Fluorescence Standards
The optical properties of an ideal fluorescence standard should be independent of the environment (solvent composition, temperature, etc.). This means, among other things, that the compound should not be involved in chemical equilibria. Alkaline solutions are to be avoided, because their alkalinity changes gradually due to absorption of carbon dioxide. Furthermore the fluorescence should not be quenched by oxygen and the fluorescence efficiency should be independent of temperature variations. The commonly used fluorescence standards fail badly on one or more of these requirements. A number of better standards can be suggested on the basis of the foregoing discussions.  Coumarin 1. This compound (7-diethylamino-4methylcoumarin) is th e most readily available 7-aminocoumarin. It is easily purified by recrystallization from ethanol. It is highly soluble in most organic solvents, but not in water. The fluorescence quantum efficiency is about 0.5 in ethanol, generally higher in less polar solvents, and is almost independent of temperature. There is little or no quenching by oxygen. A disadvantage is the possible protonation in acidic solvents. If this is a problem, Coumarin 102 or Coumarin 153 may be used. The latter compound is particularly interesting because of its large Stokesshift and the very broad fluorescence spectrum. The fluorescen ce efficien c y of Coumarin 102 was measured as 0.6 and that of Coumarin 153 as 0.4 (both in ethanol). Another compound, possibly useful as a standard, is Coumarin 6 (quantum efficiency 0.8 in ethanol), which absorbs at longer wavelengths than most other coumarin derivatives. The latter compounds are commercially available, but the price is still high. However, this should not be a deterrent, as the price will certainly come down and generally only a few milligrams are required. We feel that Coumarin 1 is superior to qumme bisulfate in every respect. It can be used in almost any solvent except water.
Rhodamine 6G-CL04• As was pointed out previously in this article, the frequently used standard Rhodamine B undergoes acid-base reactions that affect its optical properties. Its fluorescence efficiency depends strongly on type of solvent and temperature. Therefore it is not surprising that the quantum yield values reported in the literature vary considerably. As discussed above, Rhodamine 6G-perchlorate is superior to Rhodamine B, because its quantum efficiency has the value 0.95 almost independent of solvent and temperature. The dye chloride is readily available in rather pure form. It can be further purified by column chromatography on basic alumina with ethanol or methanol as the solvent. The perchlorate is insoluble in water and is easily prepared by adding HCl04 to an aqueous solution of the dye chloride. It can be recrystallized from alcohol-water mixtures. The ethyl ester perchlorate of Rhodamine 101 has the same useful properties as Rhodamine 6G-perchlorate, while absorption and fluorescence are shifted about 50 nm to the red. However, this dye is not yet commerciallyavailable.
Oxazine 170-CL04• Of all available oxazines this derivative has the highest fluorescence efficiency ( ~ 0.5 in ethanol). Its fluorescence properties are closely related to those of Rhodamine 6G-CI04. Absorption and fluorescence are shifted by about 100 nm to the red ( fig. 7). As in the case of Oxazine 4, the tluorescence efiiciency increases nearly a factor of 2 on deuteration of the amino groups. Apart from this effect, it is almost independent of solvent and temperature. However, variations of temperature have some effect on the absorption spectrum due to the annellated benzo group. In this respect Oxazine  I  \  I  I  I  I 4-CI0 4 would be preferable _ As pointed out earli er , these dyes cannot be used in basic solution_ Bein g pe rchlorates , they are practically in solubl e in water.
HexamethyLindodicarb ocyanine (HID C) and Hexa-methyLindotricarbo cyanine (HITC). Very few d yes are kn own th at a re relatively sta bl e a nd flu o resce well in th e infra red region of the spectrum. Of th e co mme rcially a vail abl e mate ri a ls, th e cya nin e d yes 1,1' ,3,3,3' ,3 ' -hexamethylind odi carbocya nin e (HIDC) iodide and 1,1 ' ,3,3,3' ,3' -hexa me th ylind otri carbocyanin e (HITC) iodide s ta nd out o n both co unts. Co ntrary to the othe r co mpound s HIDC (n=2) HITC (n=3) s ugges te d he re, the flu oresce nce e ffi cie ncy of these dyes depends markedly on type of solve nt a nd te mperature. It appears to be highest in s ulfoxides (dim e th yl s ulfoxide, te tra me th ylene s ulfoxid e). As s hown in fi gure 8, th e flu oresce nce of RITe exte nd s to 900 nm . Th e photoc he mical s tability is muc h hi gher th a n in rdated thi acarbocya nin es. A co nce ntrated so luti on of HID C-iodid e in dimethyl s ulfoxid e is c urre ntl y in use in our la borator y as a photon co unter tha t o perates up to 700 nm. It is muc h more sensitive than equ ally co nce ntra ted solutions of methylene blue as thi s has a lower flu oresce nce efficiency.
Th e flu orescence spectra and efficie ncies of the s ugges ted dyes need to be determined accurately. To us this see ms to be of greate r benefit than perpetuatin g bad sta ndard s whose only justification today is their co mmon usage.
ll OI The abso rption of Rhodam in e 6G (2g/1 in e thanol), whose max imum is at 530 nm, is still noti ceab le a t 633 nm , far beyo nd t.h e flu ores ce nce maximum of 555 n m: a 5 mW He-Ne lase r exc it es ye ll ow a nti -Stokes flu oresce nce th at ca n be observed vi s ua ll y.