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The interaction of methyl orange and other azo-dyes with polyelectrolytes and with colloidal electrolytes in dilute aqueous solution
The Interaction of Methyl Orange and Other Azo-Dyes with Polyelectrolytes and with Colloidal Electrolytes in Dilute Aqueous Solution F. Q U A D R I F O G L I O ~_~NDV. C R E S C E N Z I Istituto di Chimica, Universitd di Trieste, Trieste, Italy
Received June 15, 1970; accepted October 15, 1970 A detailed uv spectral investigation of the interaction of methyl orange (and other similar anionic azo-dyes) with four different synthetic cationic polyelectrolytes, with colloidal electrolytes, as well as with two proteins in dilute aqueous solutions, has been carried out. Under appropriate conditions, binding of methyl orange, ethyl orange, and butyl orange to poly (~-ornithine, poly-L-lysine), poly (N-methylvinyl pyridinium chloride), and trimethyldodecylammonium chloride, gives rise to marked changes in the spectrum of each of the three dyes (but not of other dyes examined). These spectral changes are quite different from those which result from the binding of the azo-dyes on to the strongly ionized poly(vinylbenzyltrimethyl ammonium chloride) chains. The effects observed with the four synthetic polyelectrolytes mentioned above and with the colloidal electrolytes, are postulated to be connected with a conformational change of bound dye molecules. Interaction of methyl orange with bovine serum albumin and with lysozyme would mainly result in a partial solubilization of the dye in the hydrophobic regions of the proteins.
INTRODUCTION Extensive studies of the interaction between a variety of cationic dyes with many different polyanions in dilute aqueous solutions have been carried out by a number of authors (I, 2). Investigations on the physicochemical behavior of anionic azo-dyes in aqueous solutions of polycations are, on the contrary, relatively scarce. A notable exception, however, is represented by the case of methyl orange. Methyl orange (R~O) has in fact been often used as a probe to study the mode and the extent of complex formation between small molecules and proteins or water-soluble polymers (3-10). The study of these complexes has given useful information about the possible type of interactions prevailing in the association between enzymes and substrates or inhibitors.
Recently Klotz et al. (10) have shown that when MO is bound to partially aeylated poly(ethylenimine) an absorption spectrum which is completely different from t h a t exhibited b y the same substance dissolved in water or in apolar solvents is observed. These authors have attributed this behavior to the stacking of M O molecules bound to the apolar aey] groups (e.g., lauroy] groups). These interesting results have prompted us to examine in some detail the spectral properties of methyl orange, ethyl orange (EO), and butyl orange (BO), and of a few other azo-dyes in aqueous solutions of synthetic cationic polyelectrolytes. Attention has been mainly directed toward elucidation of the spectral behavior of 5/[0. The following two polyelectrolytes were used in the experiments with the three dyes mentioned above: poly(L-ornithine hydrobromide) Journal of Colloid and InterfaceScience, Vol.35, No. 3, ~a~ch 1971 447
448
QUADRIFOGLIO AND CRESCENZI
(PLO) and the strongly ionized poly(vinylben~yl triethy]ammonium chloride) (PVBTEA). Absorption spectra of MO were carried out also in solutions of cationic colloidal electrolytes and in solutions of poly(L-lysine hydrobromide) (PLL), poly(N-methylvinyl pyridinium chloride) (PVP), bovine serum albumin, and lysozyme, respectively. The spectral properties of MO, as well as of EO and BO, recorded in different conditions seem to suggest that the new spectrum of the type observed by Klotz et al. (10), when MO is bound to acylated polyethylenimine under certain given conditions, might not be simply due to stacking of bound MO molecules. MATERIALS AND METHODS
Methyl orange was a C. Erba product. It was purified by repeated crystallization from ethanol. Ethyl orange and butyl orange were Eastman Organic Chemical products, used without further purification. p-Amino-azobenzene (pAAB) was synthesized according to Staedel and Bauer (11), mp 121-122°C. p-Dimethylamino-azobenzene sulfonic acid methyl ester was synthesized according to the following procedure. 1 Ten milligrams of methyl orange were dissolved in water (5 ml) and the solution was eluted through an Amberlite IRC-50 column to obtain the acid. The solution of p-dimethylamino azobenzene sulfonic acid was evaporated and then dried under vacuum. The residue was dissolved in methanol and then a solution of diazomethane in ether was added. The reaction mixture was evaporated to dryness and the residue was purified on a thin layer of silica gel using a mixture of chloroform-ethanol (95:5) as eluent. The yellow spot with Rs = 0.55 was eluted with ethanol-chloroform (50:50) and the solution evaporated to dryness. The sample obtained in this way was used without further purification. Thanks are due to Prof. G. Prota for having carried out this preparation. Journal of Colloid and Interface Science,
Vol. 35, No. 3, March 1971
Dodecylamine (DA), a BDH product, was crystallized from ethanol-water. Quaternization of poly (vinylpyridine) was carried out by addition of an excess of methyl iodide to a methanol solution of the polymer at 60°C. The reaction mixture was then refluxed overnight. The precipitated polymer was then dissolved in water and passed through an anionic exchange column in the C]- form. The polyelectrolyte was then recovered by freeze-drying. Trimethyl dodecylamine chloride (TDACl) was prepared by quaternization of dodeeylamine (DA). Methyl iodide, slightly in excess with respect to the stoichiometrie amount, was added to a DA solution in methanol and the reaction mixture was refluxed at 60°C, overnight. The precipitate was dissolved in water and then eluted through an anionic exchanger in CI- form (Dowex 1) and the TDAC1 formed was recovered by freeze-drying and then crystallized from ethanol-water. Dodecylamine hydroehloride (DAItC1) was prepared by dissolving DA in absolute ethanol and then saturating the solution with gaseous HCI. The precipitate was repeatedly crystallized from ethanol-ether (rap 169°-174°). 4-41 Hydroxy-azobehzene sulfonic acid was prepared according to Stein and Moore (12). n-Butyl-amine was obtained from C. Erba. All other solvents and products used were reagent grade. Poly(~-ornithine hydrobromide) and poly@-lysine hydrobromide) were freeze-dried Pilot, products. Bovine serum albumin (BSA) was a Sigma Chem. Corp. sample. Lysozyme was a Sigma Chem. Corp. sample, crystallized, dialyzed, and three times freeze-dried. Poly(vinylbenzyl triethylammonium chloride) PVBTEA, was a gift of Dr. Areus, ~ whose kindness is gratefully acknowledged. The absorption measurements were car2 C. L. Areus, D e p t . of Chemistry, U n i v e r s i t y of Surrey, Guilford, England.
INTERACTION
OF METHYL
ORANGE
ried out with a Hitachi Recording Spectrophotometer, Model EPS-3t, using quartz cells of different path lengths. The quartz cells were calibrated using the 370 nm peak of K2CrO4 aqueous solution at p i t = 12. Additions of solutions directly into the ceils were made using a micrometric syringe (Chemetron). pH measurements were carried out with a Radiometer pH-M4 instrument. The irradiation experiments were made using tungsten and mercury lamps. The beam of light was focused directly in quartz ceils by means of lenses. Filters were used to avoid heating of the solutions. The irradiated solutions were scanned in a period of a few seconds after removal from the light source. The conductance measurements were carried out with a Jones bridge at a frequency of 1000 Hz and with a voltage measured at
E xlO3
/
#
WITH
ELECTI~OLYTES
449
the electrodes of the cells of 4 V. As zero detector a Hewlett-Packard 130 C oscilloscope was used. The cell was immersed in a Townson and Mercer thermostat equilibrated at 25 ° ± 0.1°C. The cell used for the determination of the critical micelle concentration (e.m.c.) was equipped with bright platinum electrodes to avoid adsorption of the colloidal electrolyte. RESULTS
a. MO, EO, and BO in Aqueous Solutions. Prior to discussing the results obtained working with MO, EO, and BO in the presence of polyeleetrolytes, we wish to briefly report a few data concerning the spectral behavior of MO in water in a wide range of concentrations, up to saturation. According to these data MO does not obey Beer's law, although the deviations found in the concentration range 10-3-10 -~ M are quite small: at least
.....
-5 C= 4,1GxlO M
-
C= 1.GGxlO
-2
tj
-
M
20 /
]
1.300 0
IS
E46o
E420 1.200
10h
,'
1.100
0
~J
350
400
I
i
I
450
500
550
i -4
-3
log CMO
-2
I
GO0
~L Cnm) FIO. i. Absorption spectra of methyl orange at two different concentrations in water. In the insert the ratio between the extinction coefficients at 460 nln and 420 nm is plotted against the logarithm of the dye concentration. Journal of Colloid and Inf~rface Science, Vol, 35, No. 3, M:areh 1971
450
QUADRIFOGLIO
AND CRESCENZI
when compared with those typical of other dyes such as acridine orange, methylene blue, crystal violet (2, 13-16). The latter dyes are known to spontaneously dimerize and/or aggregate through stacking even in very dilute solutions in water, with resultant marked spectral changes. In Fig. i the spectra of MO in water at two extreme concentrations, namely, 4.16 X 10-5 and 1.66 X 10-2 M (near saturation), are reported. In the same figure the ratio ~46~/~420 against the logarithm of 5~O concentration is also plotted in order to concisely illustrate deviations from ideal spectral behavior for this dye. The data obtained working at different concentrations show that increasing concentrations of the dye bring about a blue shift. In analogy with the conclusions drawn by several authors (2, 13-16) working on other dyes we can safely assume that MO in aqueous solution shows aggregation phenomena of the type monomer dimer ~ - • • ~ polymer. The higher the aggregate, the lower is the wavelength corresponding to the absorption bands. These assumptions will also be supported by the behavior of the dye in the presence of PVBTEA. The stacking tendency of !riO is, however, much lower than that of other dyes quoted above. In addition, FIO fails to show a distinct dimer band and/or polymer bands in water solution. In the case of EO and BO we have indications that the same holds true; however, no precise measurements have been carried out because of the much lower solubility of these dyes. b. MO, EO and BO in Aqueous Solutions of PVBTEA and of PLO. The spectra recorded using dilute solutions of BO, EO, and MO, respectively, in the presence of increasing amounts of PVBTEA are reported in Fig. 2. Data of Fig. 2 indicate that early addition of the strongly ionized PVBTEA polycations results in the appearance of new absorption bands, blue-shifted with respect to those of free monomeric dye molecules in Journal of Colloid and Interface Science, Vol. 35, No. 3, March 1971
water. The new bands gradually decrease in intensity upon further additions of the polyelectrolyte. The spectra of each of the three dyes considered finally appear to revert back to the original ones, respectively, when
1.0
1
NIO
0.8
0.6
0.4
0.2
0 EO
1
121
0.4
~. o.2 o 0 B0
0.8
0.6
0.4
0.2
400
500
~, [nm)
600
FzG. 2. A b s o r p t i o n s p e c t r a of M O , E O , a n d B O i n t h e p r e s e n c e of v a r y i n g a m o u n t s of p o l y (vinyl benzyl triethylammonium chloride), pH = 6.8; 1 c m cell. NO
EO
C~O CPYBTEA CEO X 105 X 10~ (N)
1 2 3 4 5
3.77 3.76 3.75 3.71 3.67
None 2.34 4.07 6.90 22.65
BO
CPVBTEA CBO
X 105 X 105(N) X 10~
2.40 2.40 2.39 2.38 2.32
None 1.16 2.33 8.67 28.3
2.49 2.49 2.47 2.40 2.31
CPVBTEA X 10~ (N)
None 1.16 6.35 32.9 59.8
INTERACTION
OF METHYL
ORANGE
PVBTEA is in excess in the solutions. This behavior is quite similar to that shown by, for example, acridine orange upon addition of polyanions (1, 2, 15, 16). In analogy with the explanation given in the case of acridine orange and other similar dyes, the new bands may be considered as resulting from a clustering of dye molecules onto PVBTEA chains. The complexity of these clusters diminishes by successive addition of polymers as the dye is more dilute on the charged surface of the polyelectrolyte. When BO, EO, and MO can interact with an excess of PVBTEA they are bound in monomeric form to this polyelectrolyte and therefore exhibit spectral properties quite similar to those typical of the free, monomeric, state in water. The absence of isosbestic points in the series of spectra shown in Fig. 2 suggests complex equilibria between monomers, dimers, trimers and so forth. The results of the spectral measurements carried out under similar conditions for the three dyes in PLO solutions are reported in Fig. 3. In a]l spectral runs the pH of the solutions was 5-6. The interesting featm'e which immediately emerges is that for all three dyes considered, interaction with PLO brings about spectral perturbations distinctly different from those found with PVBTEA. It is seen in fact that discrete additions of PLO steadily promote the appearance of new (characteristic) sharper bands (especially in the case of I~[O) in the spectra of the dyes. These bands are more blue-shifted than those recorded under similar conditions in the presence of PVBTEA. Successive additions of PLO first increase and then decrease the amplitude of these new bands. However, whereas in the case of PVBTEA the dilution of the dye on the polyeleetrolyte is followed by a regular red shift of the absorption bands, in the case of PLO the low wavelength bands decrease but no shift of the maxima are evident. Also in the case of PLO no isosbestic points are apparent. A summary of characteristic main absorption bands of MO, EO, and BO in water and
WITH
1.0
ELECTROLYTES
451
~
NO
0.8 0.6 0.4 0.2
i N z O.6 ~ ~
0.4
~ 0.2 0 O.B I
~
BO
0.6
0.2
400
500
X (rim)
600
FIG. 3. Absorption spectra of MO, EO, and
BO in the presence of varying amounts of poly(L-ornithine hydrobromide), ptI = 5.5; 1 cm cell. MO
1 2 3 4 5
EO
BO
C~to CPLO CEO CPLO CBO CI'Lo X10S X 105 (N) X I0~ X 10~ (N) X 10~ XIO~(N) 3.76 None 2.44 None 2.49 None 3.76 2.34 2.44 2.34 2.49 1.63 3.75 14.00 2.43 4.68 2.47 8.10 3.72 46.4 2.40 69.2 2.39 33.75 3.65 156.8 2.36 158.2
in PVBTEA and PLO solutions is reported in Table I. In Fig. 4 the dependence of the extinction coefficients at 368 and 464 nm for 5~O upon PLO concentration is illustrated. This figure shows that even at PLO concentration of 10-2 N the 368 nm band for 1V[O is still clearly evident. Journal of Colloid and Interface Science,
Vol. 35, No. 3, l~areh 1971
452
QUADRIFOGL10 AND CRESCENZI TABLE I
MAIN ABSORPTION BANDS OF METHYL ORANGE (MO), ETHYL ORANGE (NO), AND BUTYL ORANGE (BO) IN WATER AND IN DILUTE AQUEOUS SOLUTION OF POLY(VINYL BENZYL TRIETHYLAMMONIUM CHLORIDE), PVBTEA, AND OF POLY(L-ORNITHINE), (PLO), RESPECTIVELY Dye
kmax (rim) in water
~am~x in PVBTEA solution
kra~x in PLO solution
MO EO BO
462 (C = 2.5 X 10-SM) 472 (C = 2.4 X 10-5 M) 480 (C = 2.5 X 10-5 M)
410 (P/D = 1.1) 415 (P/D = 1.0) 425 (P/D = 2.47
368 388 405
Approximate maximum absorption wavelength, most shifted with respect to free dye characteristic maximum absorption in water, evaluated from spectra recorded at the given P/D ratios of PVBTEA to dye concentrations (in equiv/1). The pit of the solutions was about 5.5 for PLO and about 7 for PVBTEA.
25 xlO3
20
~
~
~
~368
15 lO
S- SO
(
~
/
/
0
' -4,0
~
~464
-- -3.0 '
log CPLO
- 2,0
FzG. 4. ExtinctioB coefficient for PLO at 368 and 464 nm as a function of the logarithm of added PLO concentration. All these data invite at this stage a few comments. The new sharp band at 368 nm for MO in PLO solutions is identical to that observed b y Klotz et al. (10) working with MO in solution of partially acylated poly(ethylenimine) and attributed by these authors to bound dye stacking. 8 However in view of the results obtained with MO, as well as EO and BO, in P V B T E A For EO and BO no results of the type presented here have been reported in the literature thus far. Journal of Uolloid and Interface Science~ Vol. 35, No, 3, ~arch 1971
solutions it appears difficult to understand why dye molecules aggregated on to PLO should exhibit spectra so different from those bound to P V B T E A , if aggregation is the only relevant phenomenon. One possible explanation could be that larger dye aggregates are formed when MO, EO, and BO are bound to PLO than when these dyes are bound to P V B T E A . I n other words, exciton effects resultant from dye stacking would reach a higher value for the dye aggregates bound to P L O ; these aggregates, consequently, should comprise a
INTERACTION OF METHYL ORANGE WITH ELECTROLYTES
453
A 1.8
1.6
1.4
1.2
-
8
1.0
.8
.6
.4
.2
I
l
r
I
I
350
400
4.50
500
.550
~00
nm
650
Fio. 5. Absorption spectra of methyl orange (C = 4.4.10.5 M) in the presence of poly(L-lysine hydrobromide) (C = 1 X 10-3 N) for different additions of NaOH. Molarity of NaOH varied as follows : curve I, none; curve 2, 1.59 X 10-4; curve 3, 5.45 X 10-4; curve 4, 8.43 X 10-4; curve 5, 1.27 X 10-3; curve 6, 1.68 ?( 10-3; curve 7, 2.08 X 10-3; curve 8, 2,58 X 10-3. 1 crn cell. 5 the spectra recorded after successive additions of small amounts of concentrated N a O I t solution to the cell containing a solution of MO (C = 4.4 X 10-5 M) and P L L (1 X 10-2 equiv/1) are reported. As can be seen, reduction of charge density of the polycation b y N a O H causes a gradual reversal of the MO spectrum to the original one. The disappearance of the 368 nm band occurs with the presence of an isosbestic point and no shift of the band is detectable. The same result is obtained if one adds simple salts to solutions containing 5~O and P L L (or PLO) at p H = 5.5. I t is interesting to point out that, in this case, in order to obtain the spectrum of IV[O in the monomeric c. MO in Aqueous Solutions of Other form (probably free in solution), it is necesSynthetic Polyelectrolytes and of Colloidal sary to reach a concentration of NaC1 500 Electrolyes. A behavior analogous to that times the concentration of 5¢0. This beillustrated in Fig. 3 for 5¢O in PLO solutions havior reinforces the hypothesis that forces has been found working with poly(L-lysine other t h a n electrostatic ones stabilize the hydrobromide). With P L L we have also binding of 5¢O to polyelectrolytes like P L L studied the effect of discharging the poly- and PLO. The hydrophobie nature of these electrolyte upon the IVIO spectrum. In Fig. forces is strongly suggested b y the results greater number of interacting chromophores t h a n those bound to P V B T E A . Expectations were, however, t h a t the strongly ionized polyclectrolyte P V B T E A would be able to induce at least as much stacking of bound dyes as with PLO. Evidently, whether the stacking hypothesis is correct or not, forces other t h a n electrostatic ones a n d / o r other effects must contribute to determine the particulate state of azo-dyes bound to PLO. In view of the interest of the problem, and with the hope of contributing to its elucidation, we wish to present more data on the spectral behavior of MO in polyelectrolyte and colloidal electrolyte solutions.
Journal of Colloid and Interface Science, Vol. 35, No. 3, March 1971
454
QUADRIFOGLIO AND CRESCENZI
.8
.7
.6
B
4
.4
.3
.2
.1
o:
I
350
I
400
I
450
I
500
I
550
I
600 nm
FIG. 6. Absorption spectra of methyl orange in the presence of poly(L-ornithine hydrobromide) for different additions of dioxane. 1 cm cell. Curve C~o X 105 (M) CpLO X 105 (N) Cal..... (M) 1 2.5 1.5 None 2 2.37 1.42 0.6 3 2.16 1.29 1.39 4 2.01 1.20 2.27 obtained upon addition of aliquots of dioxane to a solution of MO and PLO. The spectra recorded after these additions are shown in Fig. 6. In this ease the lack of an isosbestic point is justified b y a continuous change in dielectric constant of the solvent mixture. A behavior similar to that shown by 5~O in the presence of poly(L-ornithine) and poly(L-lysine) was also found with another cationic polyelectrolyte, poly(vinyl-hrmethyl pyridinium chloride). This polyelectrolyte is strong enough to resist discharge upon addition of N a O H and therefore the band of MO at 368 nm in its solutions was found present in the entire range of p H from 5 to 11. Addition of NaC1 or N a O H at high concentration, however, produces the same effects as in the case of P L L and PLO. We wish, finally, to report t h a t another class of substances which can induce the new Journal of Colloid and Interface Science, ¥ol. 35, No. 3, March 1971
spectral band at ~ 3 7 0 nm of MO is that of colloidal e]ectrolytes. I t is well known that these substances in water give rise to micelles when their concentration exceeds a characteristic critical value (c.m.c). The micelles have a structure such that the polar groups of the molecules are exposed to water and the a p o l a r moieties lie in the interior to avoid contact with water. The high concentration of charge on the surface of the mice]les makes them somewhat similar to spherical polyelectrolytes. The two colloidal electrolytes used in this work are dodecylamine hydrochloride and trimethyldodecy]ammonium chloride. The c.m.c, values obtained are 1.25 X 10-2 M and 1.47 X 10-2 M for TDAC1 and DAHC1, respectively. The last value is in good agreement with the literature value (17-19). Figure 7 shows the absorption spectra of
INTERACTION OF METHYL ORANGE WITH ELECTROLYTES
455
A .15 lo
.7 9
.6
.5
.4
.3
.2
.'1
0
I
3S0
I
400
I
450
I
500
I
550
600
nm
FIG. 7. Absorption spectra of methyl orange in the presence of different amounts of dodeeylamine hydrochloride. 1 em cell. Curve C~o X 10~ (M) CI)A~c, X l0 S (M) 1 2.92 0.4 2 2.90 0.8 3 2.88 1.2 4 2.87 1.6 5 2.85 2.3 6 2.79 4.6 7 2.74 6.0 8 2.65 9.1 9 2.38 18.3 10 2.92 None MO obtained b y additions of aliquots of concentrated solutions of DAHC1 to an aqueous solution of MO at p H = 5.5. Unexpectedly the low wavelength b a n d (385 nm in this case) appears at the early additions of DAHC1. Curve 1 in Fig. 7 shows the band at 385 nm for a D A H C l concentration of 4 X 10 -4 M. Successive additions of D A H C I increase this band and decrease the absorption at 465 nm until the e.m.e, of the detergent is approached. Around this concentration the spectrum of the dye develops a b a n d at 420 nm characteristic of MO dissolved in apolar solvents. This behavior can be explained b y assuming t h a t MO is first
bound to independent D A H C I molecules or D A H C I dimers (20) and then when the micelles are formed it is solubilized in their interior. Analogous results have been obtained with the other colloidal electrolyte used, i.e., trim e t h y l d o d e c y l a m m o n i u m chloride (the low wavelength b a n d is centered at 375 n m in this case). As in the case of cationic polyelectrolytes the addition of NaCl or dioxane prior to micelle formation prevents the development of the new band. I t has to be noticed, however, that, whereas in the case of cationic polyeleetroJournal of Colloid and Interface Science, Vol. 35, No. 3, M:arch 1971
456
QUADIZIFOGLIO AND C R E S C E N Z I
lyres the dye polymer complex is indefinitely stable, in the case of dye-colloidal electrolyte complexes there is a tendency to phase separation on standing, even in the most dilute solutions. This fact has been verified by eentrifugation of the solutions followed by recording of the spectra. The results obtained with colloidal electrolytes show that the new spectral property of methyl orange is not necessarily connected with the binding to a surface with a high charge density. IIowever, experiments carried out with simpler charged amines, i.e., hexylaminc hydroehloride, trimethylamine, ethylenediamine, and lysine, at different vNues of concentration and pII, have failed to reveal the new spectral band. This result has already been obtained by Klotz (4). Experiments of the type described above for MO have been carried out also with ecrupounds similar to methyl orange, i.e. p-azobenzene sulfonic acid, p-dimethylaminoazobenzene, p-aminoazobenzene, p-dimethylaminoazobenzene methyl sulfonate, and p-hydroxyazobenzene sulfonie acid. None of these compounds, however, in water and in the presence of PLL, PLO, or colloidal electrolytes have shown relevant spectral changes in their original characteristic spectra, as opposed to the case of MO, EO, and BO. DISCUSSION
Discussion will be mainly centered on the results obtained working with l~10, as this may also help to shed some light on our (fewer) data for EO and BO. In summary our results indicate that: a) MO does not obey Beer's law. This dye, however (as well as EO and BO), has not a marked tendency to dimerize or to aggregate in water, at least when compared with other dyes, such as acridine orange, methylene blue, crystal violet. According to spectra obtained in concentrated aqueous solutions of MO, the bands corresponding to dimers or higher aggregates are located at wavelengths higher than 400 nm. b) In the presence of PVBTEA, dye moleJournal of Colloid and Interface Science, Vol. 35, No. 3, March 1971
cules bound to the polyelectrolyte, at P / D ratios lower or not much greater than unity, are certainly dimerized or aggregated (at least partially). No other explanation can be given of the blue shifts observed at the early additions of PVBTEA and of the red shift taking place at higher polymer concentrations. This behavior is quite similar to that shown by other dyes after additions of discrete amounts of polyeleetrolytes. With IViO (as well with EO and BO), on the contrary, no distinct bands corresponding to dimers or higher aggregates are detectable in the dye spectra. However, we can safely conclude that the lowest wavelength band to which corresponds the highest N[O aggregate on PVBTEA should not be located below 405 nm, approximately (see Table I for other dyes). c) In the presence of appropriate amounts of the two polyaminoacids, PLO and PLL, and in the presence of poly(N-methyl vinyl pyridinium chloride) or of cationic colloidal electrolytes at very low concentrations, 1~O exhibits a new spectrum with a relatively sharp band at ca. 370 rim. Similar results were obtained working with EO and BO in PLO solutions (see Table I). The wavelengths corresponding to these bands are always lower (as much as 25-30 rim) than the lowest wavelength at which the aggregate bands in the presence of PVBTEA may be located. As we do not see any reason why the aggregate formed on PLO, PLL, PVP should be different from those formed on PVBTEA we conclude that simple aggregation is not the complete explanation for the low wavelength bands. Increasing the concentration of PLO and P L L brings about complicating features in the spectrum of MO. In particular, in the case of PLO we have found that the intensity of the 368 nm band of MO first increases with increasing PLO concentration and subsequently decreases at P / D ratio greater than 2, without an appreciable shift in the maximum and without an isosbestie
INTERACTION
OF METHYL
ORANGE
WITH
ELECTROLYTES
4~57
point. The lack of isosbestie points denotes dimerie form to adjacent positive sites of the presence of an equilibrium between at appropriate hydrophobie moieties. Under least three absorbing forms of the dye, and this assumption one should then conclude therefore excludes the hypothesis of a dimer that MO aggregates bound to PVBTEA for the form absorbing at 368 nm. The chains are in an environment which makes hypothesis of an higher aggregate for the the 368 nm transition forbidden. This line form absorbing at 368 nm is in conflict with of reasoning applies also to BO and EO. The the persistence of the band at the same wave- other way considers the possibility that the length at different concentration of the 368 nm band depends on the conformation polyaminoaeid. In fact several works on the of the polyeation, and therefore on the availaggregation of dyes on polymer surfaces have ability of special sites of binding the number shown that high aggregates have different of which may depend on the concentration absorption wavelengths depending on the of the polymer. As to the origin of the low wavelength complexity of the aggregate and that a reduction of the statistical number of the band a tentative hypothesis may be formudye molecules of the aggregates produces a lated in terms of a eonformational transregular red shift in the spectrum (this is the formation with loss of conjugation of the behavior shown also by 1~O, BO, and EO (bound) dye molecules, e.g., a trans --+ cis when bound to PVBTEA). We think that isomerization. This transformation could be the spectra shown in Fig. 3 can be tenta- "stimulated" by a better ion-pair formation tively explained by the simultaneous pres- of the higher dipole moment form (cis) of ence of an unknown form of the dyes which MO with charged, hydrophobic sites. absorbs at 368 nm and of aggregates of This hypothesis, which we tentatively different complexity, all of them in equi- extend to the case of EO and BO bound to librium and the relative amount of which PLO, is also in agreement with the observadepends on the concentration of the polymer. tion that azoeompounds which ean be d) That the low wavelength band is not isomerized to the cis form by irradiation in simply due to stacking may be supported solution, may show bands at distinctly from the data obtained with dilute colloidal lower wavelengths than the lrans form electrolyte solutions (see Fig. 7). In this (21-25). case in fact extensive stacking would appear The cis--+ trans isomerization, furtheras consequence of the initial binding of very more, has been shown to be readily susfew, 5kely two or three, dye molecules to the ceptible to catalysis by various molecules dimers which the colloidal electrolyte may and ions (in particular by H +, OH-, and form at concentrations well below the e.m.c. NH3) in the case of photochromic dyes and Such a high stacldng tendency is not ex- photochromic macromoleeules (26). In hibited, however, by MO (point a) the view of the special ionic milieu represented aggregated species of which should not have by polyion coils domains in dilute aqueous absorbance peaks below about 405 nm in solution, a catalytic effect by pendant funcaqueous solution (points a and b). tional groups and/or their ionic-atmosphere, e) It must be pointed out nevertheless toward MO, EO, and BO isomerization, that the appearance of the 368 nm band of may not be excluded in the case of PLO, h410 in the presence of polycations is a phe- PLL, and PVP chains (and colloidal electronomenon which depends on the concentra- lytes). tion of the polyelectrolyte. This may be In addition, the hypothesis that "interexplained tentatively in two ways. One way actions (between dye and binding site assumes that the dye in order to show the and/or between neighboring bound dyes) 368 nm band must be bound in at least may enforce certain conformation of the dye Journal of Colloid and Interface Science, Vol. 35, No. 3, lVIareh 1971
458
QUADRIFOGLIO AND CRESCENZI
molecule and reinforce (increase transition the case of the colloidal electrolytes that probabilities) specific vibrational and elec- h~O prefers to be located in the interior of tronic combination in the dye molecule" the micelles instead of being bound electrohas been advanced by Bean et al. (27) in statically to their surfaces. Solubilization order to account for the so-called J band of the trans form of MO would occur in these exhibited by the dye 4,5,4',5 ~-dibenzo- cases, according to the hypothesis discussed t . . . 3,3 -dmthyl-9-methylthmcarbocyamne upon above. interaction with a variety of polyionic CONCLUSIONS macromolecules in aqueous solution. Bean Briefly summarizing, our results appear to et al. conclude that the J band of the aboveindicate that certain anionic azo-dyes of the mentioned dye arises through reaction of type considered in this work (FRO, EO, and individual dye molecules with particular BO) may interact with cationic substrates sites as a function of dye configuration and in aqueous solution according to three conformation to the site rather than being limiting mechanisms: due to dye-dye interactions in large aggre1) Solubilization in the apolar cores of gates of dye molecules. The hypothesis and conclusions of these nficelles or in the more hydrophobic regions authors seem, in our opinion, at least in part of certain biopolymers. 2) Ion-pair formation with certain catapplicable to the case of the 370 nm band of M O as discussed above. In our case, how- ionic compounds of distinctly hydrophobic ever, the trans --~ cis isomerization hypothe- character which leads to dye dimerization sis could not be directly tested as we have and/or aggregation and/or to a conformabeen unable to induce any reversible change tiona] transformation of the dyes. 3) Electrostatic binding to strongly ionin the spectrum of M 0 in water upon ized polycations which can promote dye irradiation with variable doses of uv or stacking but which does not induce any visible light. 4 conformational changes of the dyes. The last point which has to be mentioned We are of course aware that in order to is connected with the experiments carried prove the correctness and consistency of out with two proteins: bovine serum alassumptions (2) and (3), more experibumin and lysozyme. Both these proteins mental data and direct evidences are (especially lysozyme) have a certain amount necessary. Results presented here should, of basic charged groups when dissolved in however, form a useful basis for future water. These basic groups are, potentially, works. binding sites for MO. However, absorption experiments carried out with MO in soluACKNOWLEDGMENTS tions of the two proteins have failed to show This work has been sponsored by the "Consiglio any specific interaction (e.g., the 368 nm Nazionale delle Ricerche" through the "Istituto band) even in the absence of added salts. di Chimica delle Macromolecole--Ivlilano." This result can be due to an absence of REFERENCES specific sites with properties analogous to 1. See, for example, BL&KE, A., AND PEACOCKE, those along PLO and PLL chains or to a A. R., Biopolymers 6, 1225 (1968); MUKERprevalence of binding of the type already 5EE, P., AND GHOSH, A. K., J. Phys. Chem. reported by Klotz and coworkers (3-5), 67, 193 (1963); BLAtrER, G., J. Phys. Chem. 65, 1457 (1961); BARONE, G., CARAMAZZA, i.e., solubilization in the hydrophobic region R., ANn VITAGLIANO,V., Rie. Sci. 32 (II-A), of the protein. It has been shown in fact in 4 The difficulty of revealing the cis form of azocompounds in aqueous solution in irradiation experiments has been demonstrated by Brode's group (21-25). Journal of Colloid and Interface Science,
V o l . 35, N o . 3, M a r c h
1971
554 (1962); ANVFRIEVA, E. V., BIRSHWEIN, T. M., NEKRASOVA,T. N., PTITSYN, O. B., AND S~EVEr,~VA, T. V., J. Polym. Sci. 5, 358 (1968); PAL, M. K., AND BASU, S., Makroreel. Chem. 27, 69 (1958); SODA, T., AND
INTERACTION OF METHYL ORANGE WITH ELECTROLYTES
2.
3. 4. 5. 6.
7.
8. 9.
i0. 11. 12. 13.
YOSHIO~:A, K., J. Chem. Soc. Jap., 87, 324 (1966); CRESCENZI, V., QUADRIFOGLIO,F., and V. VIT.~,_GLIANO,J. Macromol. Sei., Al, 917 (1967); STRYER, L. AND BLOUT, E. R., J. Amer. Chem. Soc., 83, 1411 (1961); MoosE, J. S., PHILLIPS, G. O., PowER, D. M., AND DAVIES, J. V., J. Chem. Soc., (A), 1970, 1155, and references therein included. BRADLEY,D. F., AND WOLF, M. K., Proe. Nat. Aead. Sei. U.S. 45, 944 (1959). KLOTZ, I. M., WALKER, F. M., AND PIVAN, R. B., Y. Amer. Chem. Soc. 68, 1486 (1946). KLOTZ, I. M., J. Amer. Chem. Soe. 68, 2299 (1946). KLOTZ, I. M., AND URQUIt2~RT,J. M., J. Amer. Chem. Soe. 71, 1597 (1949). I~LOTZ,I. M., AND URQUHART,J. M., J. Amer. Chem. Soc. 71, 847 (1949). KLOTZ, I. M., BUI~KttARD, R. K., AND URQUHART, J. M., J. Amer. Chem. Soc. 74, 77 (1952). KLOTZ, I. M., AND SHIKAMA, 1~., Arch. Biochem. Biophys. 19.3, 551 (1968). KLOTZ, I. M., AND SLONIEWSKY, A. R., Biochem. Biophys. Res. Commun. 31, 421 (1968). KLOTZ, I. M., ROYER, G. P., ANn SLONIEWSKY, A. R., Biochemistry, 8, 4752 (1969). STAEDEL,W., AND BAUER, H. Ber. Bunsenges. Phys. Chem. 19, 1952 (1886). STEIN, H., AND MOORE, G. Biochem. Prep., 1, 7 (1949). R.%BINOVITCft, E., AND EPSTEIN, L. F., J. Amer. Chem. Soc. 63, 69 (1941).
459
14. ZANKER, V., Z. Phys. Chem. (Leipzig) 199, 225 (1952). 15. LAMIN, M. E., AND NEVILLE, D. M., JR., J. Phys. Chem. 69, 3872 (1965). 16. BARONE, G., COSTzkNTINO, L., AND VITAGLI.A-ANO, V., Rie. Sci. 34 (II-A), 67 (1964). 17. RALSTON, A. W., AND ttOEaa, C. W., J. Amer. Chem. Soc. 64, 772 (1942). 18. HHARKINS,G. "The Physical Chemistry of Surface Films," p. 304. Reinhold, New York, 1957. 19. BOTR~, C., CRESCENZI, V., AND LIQUORI, A. M., "Original Lectures 3rd International Congress of Surface Activity," Cologne, p 302 (1960). 20. HIsI
Journal of Colloid and Interface Science, Vol. 35, No. 3, March 1971
Received June 15, 1970; accepted October 15, 1970 A detailed uv spectral investigation of the interaction of methyl orange (and other similar anionic azo-dyes) with four different synthetic cationic polyelectrolytes, with colloidal electrolytes, as well as with two proteins in dilute aqueous solutions, has been carried out. Under appropriate conditions, binding of methyl orange, ethyl orange, and butyl orange to poly (~-ornithine, poly-L-lysine), poly (N-methylvinyl pyridinium chloride), and trimethyldodecylammonium chloride, gives rise to marked changes in the spectrum of each of the three dyes (but not of other dyes examined). These spectral changes are quite different from those which result from the binding of the azo-dyes on to the strongly ionized poly(vinylbenzyltrimethyl ammonium chloride) chains. The effects observed with the four synthetic polyelectrolytes mentioned above and with the colloidal electrolytes, are postulated to be connected with a conformational change of bound dye molecules. Interaction of methyl orange with bovine serum albumin and with lysozyme would mainly result in a partial solubilization of the dye in the hydrophobic regions of the proteins.
INTRODUCTION Extensive studies of the interaction between a variety of cationic dyes with many different polyanions in dilute aqueous solutions have been carried out by a number of authors (I, 2). Investigations on the physicochemical behavior of anionic azo-dyes in aqueous solutions of polycations are, on the contrary, relatively scarce. A notable exception, however, is represented by the case of methyl orange. Methyl orange (R~O) has in fact been often used as a probe to study the mode and the extent of complex formation between small molecules and proteins or water-soluble polymers (3-10). The study of these complexes has given useful information about the possible type of interactions prevailing in the association between enzymes and substrates or inhibitors.
Recently Klotz et al. (10) have shown that when MO is bound to partially aeylated poly(ethylenimine) an absorption spectrum which is completely different from t h a t exhibited b y the same substance dissolved in water or in apolar solvents is observed. These authors have attributed this behavior to the stacking of M O molecules bound to the apolar aey] groups (e.g., lauroy] groups). These interesting results have prompted us to examine in some detail the spectral properties of methyl orange, ethyl orange (EO), and butyl orange (BO), and of a few other azo-dyes in aqueous solutions of synthetic cationic polyelectrolytes. Attention has been mainly directed toward elucidation of the spectral behavior of 5/[0. The following two polyelectrolytes were used in the experiments with the three dyes mentioned above: poly(L-ornithine hydrobromide) Journal of Colloid and InterfaceScience, Vol.35, No. 3, ~a~ch 1971 447
448
QUADRIFOGLIO AND CRESCENZI
(PLO) and the strongly ionized poly(vinylben~yl triethy]ammonium chloride) (PVBTEA). Absorption spectra of MO were carried out also in solutions of cationic colloidal electrolytes and in solutions of poly(L-lysine hydrobromide) (PLL), poly(N-methylvinyl pyridinium chloride) (PVP), bovine serum albumin, and lysozyme, respectively. The spectral properties of MO, as well as of EO and BO, recorded in different conditions seem to suggest that the new spectrum of the type observed by Klotz et al. (10), when MO is bound to acylated polyethylenimine under certain given conditions, might not be simply due to stacking of bound MO molecules. MATERIALS AND METHODS
Methyl orange was a C. Erba product. It was purified by repeated crystallization from ethanol. Ethyl orange and butyl orange were Eastman Organic Chemical products, used without further purification. p-Amino-azobenzene (pAAB) was synthesized according to Staedel and Bauer (11), mp 121-122°C. p-Dimethylamino-azobenzene sulfonic acid methyl ester was synthesized according to the following procedure. 1 Ten milligrams of methyl orange were dissolved in water (5 ml) and the solution was eluted through an Amberlite IRC-50 column to obtain the acid. The solution of p-dimethylamino azobenzene sulfonic acid was evaporated and then dried under vacuum. The residue was dissolved in methanol and then a solution of diazomethane in ether was added. The reaction mixture was evaporated to dryness and the residue was purified on a thin layer of silica gel using a mixture of chloroform-ethanol (95:5) as eluent. The yellow spot with Rs = 0.55 was eluted with ethanol-chloroform (50:50) and the solution evaporated to dryness. The sample obtained in this way was used without further purification. Thanks are due to Prof. G. Prota for having carried out this preparation. Journal of Colloid and Interface Science,
Vol. 35, No. 3, March 1971
Dodecylamine (DA), a BDH product, was crystallized from ethanol-water. Quaternization of poly (vinylpyridine) was carried out by addition of an excess of methyl iodide to a methanol solution of the polymer at 60°C. The reaction mixture was then refluxed overnight. The precipitated polymer was then dissolved in water and passed through an anionic exchange column in the C]- form. The polyelectrolyte was then recovered by freeze-drying. Trimethyl dodecylamine chloride (TDACl) was prepared by quaternization of dodeeylamine (DA). Methyl iodide, slightly in excess with respect to the stoichiometrie amount, was added to a DA solution in methanol and the reaction mixture was refluxed at 60°C, overnight. The precipitate was dissolved in water and then eluted through an anionic exchanger in CI- form (Dowex 1) and the TDAC1 formed was recovered by freeze-drying and then crystallized from ethanol-water. Dodecylamine hydroehloride (DAItC1) was prepared by dissolving DA in absolute ethanol and then saturating the solution with gaseous HCI. The precipitate was repeatedly crystallized from ethanol-ether (rap 169°-174°). 4-41 Hydroxy-azobehzene sulfonic acid was prepared according to Stein and Moore (12). n-Butyl-amine was obtained from C. Erba. All other solvents and products used were reagent grade. Poly(~-ornithine hydrobromide) and poly@-lysine hydrobromide) were freeze-dried Pilot, products. Bovine serum albumin (BSA) was a Sigma Chem. Corp. sample. Lysozyme was a Sigma Chem. Corp. sample, crystallized, dialyzed, and three times freeze-dried. Poly(vinylbenzyl triethylammonium chloride) PVBTEA, was a gift of Dr. Areus, ~ whose kindness is gratefully acknowledged. The absorption measurements were car2 C. L. Areus, D e p t . of Chemistry, U n i v e r s i t y of Surrey, Guilford, England.
INTERACTION
OF METHYL
ORANGE
ried out with a Hitachi Recording Spectrophotometer, Model EPS-3t, using quartz cells of different path lengths. The quartz cells were calibrated using the 370 nm peak of K2CrO4 aqueous solution at p i t = 12. Additions of solutions directly into the ceils were made using a micrometric syringe (Chemetron). pH measurements were carried out with a Radiometer pH-M4 instrument. The irradiation experiments were made using tungsten and mercury lamps. The beam of light was focused directly in quartz ceils by means of lenses. Filters were used to avoid heating of the solutions. The irradiated solutions were scanned in a period of a few seconds after removal from the light source. The conductance measurements were carried out with a Jones bridge at a frequency of 1000 Hz and with a voltage measured at
E xlO3
/
#
WITH
ELECTI~OLYTES
449
the electrodes of the cells of 4 V. As zero detector a Hewlett-Packard 130 C oscilloscope was used. The cell was immersed in a Townson and Mercer thermostat equilibrated at 25 ° ± 0.1°C. The cell used for the determination of the critical micelle concentration (e.m.c.) was equipped with bright platinum electrodes to avoid adsorption of the colloidal electrolyte. RESULTS
a. MO, EO, and BO in Aqueous Solutions. Prior to discussing the results obtained working with MO, EO, and BO in the presence of polyeleetrolytes, we wish to briefly report a few data concerning the spectral behavior of MO in water in a wide range of concentrations, up to saturation. According to these data MO does not obey Beer's law, although the deviations found in the concentration range 10-3-10 -~ M are quite small: at least
.....
-5 C= 4,1GxlO M
-
C= 1.GGxlO
-2
tj
-
M
20 /
]
1.300 0
IS
E46o
E420 1.200
10h
,'
1.100
0
~J
350
400
I
i
I
450
500
550
i -4
-3
log CMO
-2
I
GO0
~L Cnm) FIO. i. Absorption spectra of methyl orange at two different concentrations in water. In the insert the ratio between the extinction coefficients at 460 nln and 420 nm is plotted against the logarithm of the dye concentration. Journal of Colloid and Inf~rface Science, Vol, 35, No. 3, M:areh 1971
450
QUADRIFOGLIO
AND CRESCENZI
when compared with those typical of other dyes such as acridine orange, methylene blue, crystal violet (2, 13-16). The latter dyes are known to spontaneously dimerize and/or aggregate through stacking even in very dilute solutions in water, with resultant marked spectral changes. In Fig. i the spectra of MO in water at two extreme concentrations, namely, 4.16 X 10-5 and 1.66 X 10-2 M (near saturation), are reported. In the same figure the ratio ~46~/~420 against the logarithm of 5~O concentration is also plotted in order to concisely illustrate deviations from ideal spectral behavior for this dye. The data obtained working at different concentrations show that increasing concentrations of the dye bring about a blue shift. In analogy with the conclusions drawn by several authors (2, 13-16) working on other dyes we can safely assume that MO in aqueous solution shows aggregation phenomena of the type monomer dimer ~ - • • ~ polymer. The higher the aggregate, the lower is the wavelength corresponding to the absorption bands. These assumptions will also be supported by the behavior of the dye in the presence of PVBTEA. The stacking tendency of !riO is, however, much lower than that of other dyes quoted above. In addition, FIO fails to show a distinct dimer band and/or polymer bands in water solution. In the case of EO and BO we have indications that the same holds true; however, no precise measurements have been carried out because of the much lower solubility of these dyes. b. MO, EO and BO in Aqueous Solutions of PVBTEA and of PLO. The spectra recorded using dilute solutions of BO, EO, and MO, respectively, in the presence of increasing amounts of PVBTEA are reported in Fig. 2. Data of Fig. 2 indicate that early addition of the strongly ionized PVBTEA polycations results in the appearance of new absorption bands, blue-shifted with respect to those of free monomeric dye molecules in Journal of Colloid and Interface Science, Vol. 35, No. 3, March 1971
water. The new bands gradually decrease in intensity upon further additions of the polyelectrolyte. The spectra of each of the three dyes considered finally appear to revert back to the original ones, respectively, when
1.0
1
NIO
0.8
0.6
0.4
0.2
0 EO
1
121
0.4
~. o.2 o 0 B0
0.8
0.6
0.4
0.2
400
500
~, [nm)
600
FzG. 2. A b s o r p t i o n s p e c t r a of M O , E O , a n d B O i n t h e p r e s e n c e of v a r y i n g a m o u n t s of p o l y (vinyl benzyl triethylammonium chloride), pH = 6.8; 1 c m cell. NO
EO
C~O CPYBTEA CEO X 105 X 10~ (N)
1 2 3 4 5
3.77 3.76 3.75 3.71 3.67
None 2.34 4.07 6.90 22.65
BO
CPVBTEA CBO
X 105 X 105(N) X 10~
2.40 2.40 2.39 2.38 2.32
None 1.16 2.33 8.67 28.3
2.49 2.49 2.47 2.40 2.31
CPVBTEA X 10~ (N)
None 1.16 6.35 32.9 59.8
INTERACTION
OF METHYL
ORANGE
PVBTEA is in excess in the solutions. This behavior is quite similar to that shown by, for example, acridine orange upon addition of polyanions (1, 2, 15, 16). In analogy with the explanation given in the case of acridine orange and other similar dyes, the new bands may be considered as resulting from a clustering of dye molecules onto PVBTEA chains. The complexity of these clusters diminishes by successive addition of polymers as the dye is more dilute on the charged surface of the polyelectrolyte. When BO, EO, and MO can interact with an excess of PVBTEA they are bound in monomeric form to this polyelectrolyte and therefore exhibit spectral properties quite similar to those typical of the free, monomeric, state in water. The absence of isosbestic points in the series of spectra shown in Fig. 2 suggests complex equilibria between monomers, dimers, trimers and so forth. The results of the spectral measurements carried out under similar conditions for the three dyes in PLO solutions are reported in Fig. 3. In a]l spectral runs the pH of the solutions was 5-6. The interesting featm'e which immediately emerges is that for all three dyes considered, interaction with PLO brings about spectral perturbations distinctly different from those found with PVBTEA. It is seen in fact that discrete additions of PLO steadily promote the appearance of new (characteristic) sharper bands (especially in the case of I~[O) in the spectra of the dyes. These bands are more blue-shifted than those recorded under similar conditions in the presence of PVBTEA. Successive additions of PLO first increase and then decrease the amplitude of these new bands. However, whereas in the case of PVBTEA the dilution of the dye on the polyeleetrolyte is followed by a regular red shift of the absorption bands, in the case of PLO the low wavelength bands decrease but no shift of the maxima are evident. Also in the case of PLO no isosbestic points are apparent. A summary of characteristic main absorption bands of MO, EO, and BO in water and
WITH
1.0
ELECTROLYTES
451
~
NO
0.8 0.6 0.4 0.2
i N z O.6 ~ ~
0.4
~ 0.2 0 O.B I
~
BO
0.6
0.2
400
500
X (rim)
600
FIG. 3. Absorption spectra of MO, EO, and
BO in the presence of varying amounts of poly(L-ornithine hydrobromide), ptI = 5.5; 1 cm cell. MO
1 2 3 4 5
EO
BO
C~to CPLO CEO CPLO CBO CI'Lo X10S X 105 (N) X I0~ X 10~ (N) X 10~ XIO~(N) 3.76 None 2.44 None 2.49 None 3.76 2.34 2.44 2.34 2.49 1.63 3.75 14.00 2.43 4.68 2.47 8.10 3.72 46.4 2.40 69.2 2.39 33.75 3.65 156.8 2.36 158.2
in PVBTEA and PLO solutions is reported in Table I. In Fig. 4 the dependence of the extinction coefficients at 368 and 464 nm for 5~O upon PLO concentration is illustrated. This figure shows that even at PLO concentration of 10-2 N the 368 nm band for 1V[O is still clearly evident. Journal of Colloid and Interface Science,
Vol. 35, No. 3, l~areh 1971
452
QUADRIFOGL10 AND CRESCENZI TABLE I
MAIN ABSORPTION BANDS OF METHYL ORANGE (MO), ETHYL ORANGE (NO), AND BUTYL ORANGE (BO) IN WATER AND IN DILUTE AQUEOUS SOLUTION OF POLY(VINYL BENZYL TRIETHYLAMMONIUM CHLORIDE), PVBTEA, AND OF POLY(L-ORNITHINE), (PLO), RESPECTIVELY Dye
kmax (rim) in water
~am~x in PVBTEA solution
kra~x in PLO solution
MO EO BO
462 (C = 2.5 X 10-SM) 472 (C = 2.4 X 10-5 M) 480 (C = 2.5 X 10-5 M)
410 (P/D = 1.1) 415 (P/D = 1.0) 425 (P/D = 2.47
368 388 405
Approximate maximum absorption wavelength, most shifted with respect to free dye characteristic maximum absorption in water, evaluated from spectra recorded at the given P/D ratios of PVBTEA to dye concentrations (in equiv/1). The pit of the solutions was about 5.5 for PLO and about 7 for PVBTEA.
25 xlO3
20
~
~
~
~368
15 lO
S- SO
(
~
/
/
0
' -4,0
~
~464
-- -3.0 '
log CPLO
- 2,0
FzG. 4. ExtinctioB coefficient for PLO at 368 and 464 nm as a function of the logarithm of added PLO concentration. All these data invite at this stage a few comments. The new sharp band at 368 nm for MO in PLO solutions is identical to that observed b y Klotz et al. (10) working with MO in solution of partially acylated poly(ethylenimine) and attributed by these authors to bound dye stacking. 8 However in view of the results obtained with MO, as well as EO and BO, in P V B T E A For EO and BO no results of the type presented here have been reported in the literature thus far. Journal of Uolloid and Interface Science~ Vol. 35, No, 3, ~arch 1971
solutions it appears difficult to understand why dye molecules aggregated on to PLO should exhibit spectra so different from those bound to P V B T E A , if aggregation is the only relevant phenomenon. One possible explanation could be that larger dye aggregates are formed when MO, EO, and BO are bound to PLO than when these dyes are bound to P V B T E A . I n other words, exciton effects resultant from dye stacking would reach a higher value for the dye aggregates bound to P L O ; these aggregates, consequently, should comprise a
INTERACTION OF METHYL ORANGE WITH ELECTROLYTES
453
A 1.8
1.6
1.4
1.2
-
8
1.0
.8
.6
.4
.2
I
l
r
I
I
350
400
4.50
500
.550
~00
nm
650
Fio. 5. Absorption spectra of methyl orange (C = 4.4.10.5 M) in the presence of poly(L-lysine hydrobromide) (C = 1 X 10-3 N) for different additions of NaOH. Molarity of NaOH varied as follows : curve I, none; curve 2, 1.59 X 10-4; curve 3, 5.45 X 10-4; curve 4, 8.43 X 10-4; curve 5, 1.27 X 10-3; curve 6, 1.68 ?( 10-3; curve 7, 2.08 X 10-3; curve 8, 2,58 X 10-3. 1 crn cell. 5 the spectra recorded after successive additions of small amounts of concentrated N a O I t solution to the cell containing a solution of MO (C = 4.4 X 10-5 M) and P L L (1 X 10-2 equiv/1) are reported. As can be seen, reduction of charge density of the polycation b y N a O H causes a gradual reversal of the MO spectrum to the original one. The disappearance of the 368 nm band occurs with the presence of an isosbestic point and no shift of the band is detectable. The same result is obtained if one adds simple salts to solutions containing 5~O and P L L (or PLO) at p H = 5.5. I t is interesting to point out that, in this case, in order to obtain the spectrum of IV[O in the monomeric c. MO in Aqueous Solutions of Other form (probably free in solution), it is necesSynthetic Polyelectrolytes and of Colloidal sary to reach a concentration of NaC1 500 Electrolyes. A behavior analogous to that times the concentration of 5¢0. This beillustrated in Fig. 3 for 5¢O in PLO solutions havior reinforces the hypothesis that forces has been found working with poly(L-lysine other t h a n electrostatic ones stabilize the hydrobromide). With P L L we have also binding of 5¢O to polyelectrolytes like P L L studied the effect of discharging the poly- and PLO. The hydrophobie nature of these electrolyte upon the IVIO spectrum. In Fig. forces is strongly suggested b y the results greater number of interacting chromophores t h a n those bound to P V B T E A . Expectations were, however, t h a t the strongly ionized polyclectrolyte P V B T E A would be able to induce at least as much stacking of bound dyes as with PLO. Evidently, whether the stacking hypothesis is correct or not, forces other t h a n electrostatic ones a n d / o r other effects must contribute to determine the particulate state of azo-dyes bound to PLO. In view of the interest of the problem, and with the hope of contributing to its elucidation, we wish to present more data on the spectral behavior of MO in polyelectrolyte and colloidal electrolyte solutions.
Journal of Colloid and Interface Science, Vol. 35, No. 3, March 1971
454
QUADRIFOGLIO AND CRESCENZI
.8
.7
.6
B
4
.4
.3
.2
.1
o:
I
350
I
400
I
450
I
500
I
550
I
600 nm
FIG. 6. Absorption spectra of methyl orange in the presence of poly(L-ornithine hydrobromide) for different additions of dioxane. 1 cm cell. Curve C~o X 105 (M) CpLO X 105 (N) Cal..... (M) 1 2.5 1.5 None 2 2.37 1.42 0.6 3 2.16 1.29 1.39 4 2.01 1.20 2.27 obtained upon addition of aliquots of dioxane to a solution of MO and PLO. The spectra recorded after these additions are shown in Fig. 6. In this ease the lack of an isosbestic point is justified b y a continuous change in dielectric constant of the solvent mixture. A behavior similar to that shown by 5~O in the presence of poly(L-ornithine) and poly(L-lysine) was also found with another cationic polyelectrolyte, poly(vinyl-hrmethyl pyridinium chloride). This polyelectrolyte is strong enough to resist discharge upon addition of N a O H and therefore the band of MO at 368 nm in its solutions was found present in the entire range of p H from 5 to 11. Addition of NaC1 or N a O H at high concentration, however, produces the same effects as in the case of P L L and PLO. We wish, finally, to report t h a t another class of substances which can induce the new Journal of Colloid and Interface Science, ¥ol. 35, No. 3, March 1971
spectral band at ~ 3 7 0 nm of MO is that of colloidal e]ectrolytes. I t is well known that these substances in water give rise to micelles when their concentration exceeds a characteristic critical value (c.m.c). The micelles have a structure such that the polar groups of the molecules are exposed to water and the a p o l a r moieties lie in the interior to avoid contact with water. The high concentration of charge on the surface of the mice]les makes them somewhat similar to spherical polyelectrolytes. The two colloidal electrolytes used in this work are dodecylamine hydrochloride and trimethyldodecy]ammonium chloride. The c.m.c, values obtained are 1.25 X 10-2 M and 1.47 X 10-2 M for TDAC1 and DAHC1, respectively. The last value is in good agreement with the literature value (17-19). Figure 7 shows the absorption spectra of
INTERACTION OF METHYL ORANGE WITH ELECTROLYTES
455
A .15 lo
.7 9
.6
.5
.4
.3
.2
.'1
0
I
3S0
I
400
I
450
I
500
I
550
600
nm
FIG. 7. Absorption spectra of methyl orange in the presence of different amounts of dodeeylamine hydrochloride. 1 em cell. Curve C~o X 10~ (M) CI)A~c, X l0 S (M) 1 2.92 0.4 2 2.90 0.8 3 2.88 1.2 4 2.87 1.6 5 2.85 2.3 6 2.79 4.6 7 2.74 6.0 8 2.65 9.1 9 2.38 18.3 10 2.92 None MO obtained b y additions of aliquots of concentrated solutions of DAHC1 to an aqueous solution of MO at p H = 5.5. Unexpectedly the low wavelength b a n d (385 nm in this case) appears at the early additions of DAHC1. Curve 1 in Fig. 7 shows the band at 385 nm for a D A H C l concentration of 4 X 10 -4 M. Successive additions of D A H C I increase this band and decrease the absorption at 465 nm until the e.m.e, of the detergent is approached. Around this concentration the spectrum of the dye develops a b a n d at 420 nm characteristic of MO dissolved in apolar solvents. This behavior can be explained b y assuming t h a t MO is first
bound to independent D A H C I molecules or D A H C I dimers (20) and then when the micelles are formed it is solubilized in their interior. Analogous results have been obtained with the other colloidal electrolyte used, i.e., trim e t h y l d o d e c y l a m m o n i u m chloride (the low wavelength b a n d is centered at 375 n m in this case). As in the case of cationic polyelectrolytes the addition of NaCl or dioxane prior to micelle formation prevents the development of the new band. I t has to be noticed, however, that, whereas in the case of cationic polyeleetroJournal of Colloid and Interface Science, Vol. 35, No. 3, M:arch 1971
456
QUADIZIFOGLIO AND C R E S C E N Z I
lyres the dye polymer complex is indefinitely stable, in the case of dye-colloidal electrolyte complexes there is a tendency to phase separation on standing, even in the most dilute solutions. This fact has been verified by eentrifugation of the solutions followed by recording of the spectra. The results obtained with colloidal electrolytes show that the new spectral property of methyl orange is not necessarily connected with the binding to a surface with a high charge density. IIowever, experiments carried out with simpler charged amines, i.e., hexylaminc hydroehloride, trimethylamine, ethylenediamine, and lysine, at different vNues of concentration and pII, have failed to reveal the new spectral band. This result has already been obtained by Klotz (4). Experiments of the type described above for MO have been carried out also with ecrupounds similar to methyl orange, i.e. p-azobenzene sulfonic acid, p-dimethylaminoazobenzene, p-aminoazobenzene, p-dimethylaminoazobenzene methyl sulfonate, and p-hydroxyazobenzene sulfonie acid. None of these compounds, however, in water and in the presence of PLL, PLO, or colloidal electrolytes have shown relevant spectral changes in their original characteristic spectra, as opposed to the case of MO, EO, and BO. DISCUSSION
Discussion will be mainly centered on the results obtained working with l~10, as this may also help to shed some light on our (fewer) data for EO and BO. In summary our results indicate that: a) MO does not obey Beer's law. This dye, however (as well as EO and BO), has not a marked tendency to dimerize or to aggregate in water, at least when compared with other dyes, such as acridine orange, methylene blue, crystal violet. According to spectra obtained in concentrated aqueous solutions of MO, the bands corresponding to dimers or higher aggregates are located at wavelengths higher than 400 nm. b) In the presence of PVBTEA, dye moleJournal of Colloid and Interface Science, Vol. 35, No. 3, March 1971
cules bound to the polyelectrolyte, at P / D ratios lower or not much greater than unity, are certainly dimerized or aggregated (at least partially). No other explanation can be given of the blue shifts observed at the early additions of PVBTEA and of the red shift taking place at higher polymer concentrations. This behavior is quite similar to that shown by other dyes after additions of discrete amounts of polyeleetrolytes. With IViO (as well with EO and BO), on the contrary, no distinct bands corresponding to dimers or higher aggregates are detectable in the dye spectra. However, we can safely conclude that the lowest wavelength band to which corresponds the highest N[O aggregate on PVBTEA should not be located below 405 nm, approximately (see Table I for other dyes). c) In the presence of appropriate amounts of the two polyaminoacids, PLO and PLL, and in the presence of poly(N-methyl vinyl pyridinium chloride) or of cationic colloidal electrolytes at very low concentrations, 1~O exhibits a new spectrum with a relatively sharp band at ca. 370 rim. Similar results were obtained working with EO and BO in PLO solutions (see Table I). The wavelengths corresponding to these bands are always lower (as much as 25-30 rim) than the lowest wavelength at which the aggregate bands in the presence of PVBTEA may be located. As we do not see any reason why the aggregate formed on PLO, PLL, PVP should be different from those formed on PVBTEA we conclude that simple aggregation is not the complete explanation for the low wavelength bands. Increasing the concentration of PLO and P L L brings about complicating features in the spectrum of MO. In particular, in the case of PLO we have found that the intensity of the 368 nm band of MO first increases with increasing PLO concentration and subsequently decreases at P / D ratio greater than 2, without an appreciable shift in the maximum and without an isosbestie
INTERACTION
OF METHYL
ORANGE
WITH
ELECTROLYTES
4~57
point. The lack of isosbestie points denotes dimerie form to adjacent positive sites of the presence of an equilibrium between at appropriate hydrophobie moieties. Under least three absorbing forms of the dye, and this assumption one should then conclude therefore excludes the hypothesis of a dimer that MO aggregates bound to PVBTEA for the form absorbing at 368 nm. The chains are in an environment which makes hypothesis of an higher aggregate for the the 368 nm transition forbidden. This line form absorbing at 368 nm is in conflict with of reasoning applies also to BO and EO. The the persistence of the band at the same wave- other way considers the possibility that the length at different concentration of the 368 nm band depends on the conformation polyaminoaeid. In fact several works on the of the polyeation, and therefore on the availaggregation of dyes on polymer surfaces have ability of special sites of binding the number shown that high aggregates have different of which may depend on the concentration absorption wavelengths depending on the of the polymer. As to the origin of the low wavelength complexity of the aggregate and that a reduction of the statistical number of the band a tentative hypothesis may be formudye molecules of the aggregates produces a lated in terms of a eonformational transregular red shift in the spectrum (this is the formation with loss of conjugation of the behavior shown also by 1~O, BO, and EO (bound) dye molecules, e.g., a trans --+ cis when bound to PVBTEA). We think that isomerization. This transformation could be the spectra shown in Fig. 3 can be tenta- "stimulated" by a better ion-pair formation tively explained by the simultaneous pres- of the higher dipole moment form (cis) of ence of an unknown form of the dyes which MO with charged, hydrophobic sites. absorbs at 368 nm and of aggregates of This hypothesis, which we tentatively different complexity, all of them in equi- extend to the case of EO and BO bound to librium and the relative amount of which PLO, is also in agreement with the observadepends on the concentration of the polymer. tion that azoeompounds which ean be d) That the low wavelength band is not isomerized to the cis form by irradiation in simply due to stacking may be supported solution, may show bands at distinctly from the data obtained with dilute colloidal lower wavelengths than the lrans form electrolyte solutions (see Fig. 7). In this (21-25). case in fact extensive stacking would appear The cis--+ trans isomerization, furtheras consequence of the initial binding of very more, has been shown to be readily susfew, 5kely two or three, dye molecules to the ceptible to catalysis by various molecules dimers which the colloidal electrolyte may and ions (in particular by H +, OH-, and form at concentrations well below the e.m.c. NH3) in the case of photochromic dyes and Such a high stacldng tendency is not ex- photochromic macromoleeules (26). In hibited, however, by MO (point a) the view of the special ionic milieu represented aggregated species of which should not have by polyion coils domains in dilute aqueous absorbance peaks below about 405 nm in solution, a catalytic effect by pendant funcaqueous solution (points a and b). tional groups and/or their ionic-atmosphere, e) It must be pointed out nevertheless toward MO, EO, and BO isomerization, that the appearance of the 368 nm band of may not be excluded in the case of PLO, h410 in the presence of polycations is a phe- PLL, and PVP chains (and colloidal electronomenon which depends on the concentra- lytes). tion of the polyelectrolyte. This may be In addition, the hypothesis that "interexplained tentatively in two ways. One way actions (between dye and binding site assumes that the dye in order to show the and/or between neighboring bound dyes) 368 nm band must be bound in at least may enforce certain conformation of the dye Journal of Colloid and Interface Science, Vol. 35, No. 3, lVIareh 1971
458
QUADRIFOGLIO AND CRESCENZI
molecule and reinforce (increase transition the case of the colloidal electrolytes that probabilities) specific vibrational and elec- h~O prefers to be located in the interior of tronic combination in the dye molecule" the micelles instead of being bound electrohas been advanced by Bean et al. (27) in statically to their surfaces. Solubilization order to account for the so-called J band of the trans form of MO would occur in these exhibited by the dye 4,5,4',5 ~-dibenzo- cases, according to the hypothesis discussed t . . . 3,3 -dmthyl-9-methylthmcarbocyamne upon above. interaction with a variety of polyionic CONCLUSIONS macromolecules in aqueous solution. Bean Briefly summarizing, our results appear to et al. conclude that the J band of the aboveindicate that certain anionic azo-dyes of the mentioned dye arises through reaction of type considered in this work (FRO, EO, and individual dye molecules with particular BO) may interact with cationic substrates sites as a function of dye configuration and in aqueous solution according to three conformation to the site rather than being limiting mechanisms: due to dye-dye interactions in large aggre1) Solubilization in the apolar cores of gates of dye molecules. The hypothesis and conclusions of these nficelles or in the more hydrophobic regions authors seem, in our opinion, at least in part of certain biopolymers. 2) Ion-pair formation with certain catapplicable to the case of the 370 nm band of M O as discussed above. In our case, how- ionic compounds of distinctly hydrophobic ever, the trans --~ cis isomerization hypothe- character which leads to dye dimerization sis could not be directly tested as we have and/or aggregation and/or to a conformabeen unable to induce any reversible change tiona] transformation of the dyes. 3) Electrostatic binding to strongly ionin the spectrum of M 0 in water upon ized polycations which can promote dye irradiation with variable doses of uv or stacking but which does not induce any visible light. 4 conformational changes of the dyes. The last point which has to be mentioned We are of course aware that in order to is connected with the experiments carried prove the correctness and consistency of out with two proteins: bovine serum alassumptions (2) and (3), more experibumin and lysozyme. Both these proteins mental data and direct evidences are (especially lysozyme) have a certain amount necessary. Results presented here should, of basic charged groups when dissolved in however, form a useful basis for future water. These basic groups are, potentially, works. binding sites for MO. However, absorption experiments carried out with MO in soluACKNOWLEDGMENTS tions of the two proteins have failed to show This work has been sponsored by the "Consiglio any specific interaction (e.g., the 368 nm Nazionale delle Ricerche" through the "Istituto band) even in the absence of added salts. di Chimica delle Macromolecole--Ivlilano." This result can be due to an absence of REFERENCES specific sites with properties analogous to 1. See, for example, BL&KE, A., AND PEACOCKE, those along PLO and PLL chains or to a A. R., Biopolymers 6, 1225 (1968); MUKERprevalence of binding of the type already 5EE, P., AND GHOSH, A. K., J. Phys. Chem. reported by Klotz and coworkers (3-5), 67, 193 (1963); BLAtrER, G., J. Phys. Chem. 65, 1457 (1961); BARONE, G., CARAMAZZA, i.e., solubilization in the hydrophobic region R., ANn VITAGLIANO,V., Rie. Sci. 32 (II-A), of the protein. It has been shown in fact in 4 The difficulty of revealing the cis form of azocompounds in aqueous solution in irradiation experiments has been demonstrated by Brode's group (21-25). Journal of Colloid and Interface Science,
V o l . 35, N o . 3, M a r c h
1971
554 (1962); ANVFRIEVA, E. V., BIRSHWEIN, T. M., NEKRASOVA,T. N., PTITSYN, O. B., AND S~EVEr,~VA, T. V., J. Polym. Sci. 5, 358 (1968); PAL, M. K., AND BASU, S., Makroreel. Chem. 27, 69 (1958); SODA, T., AND
INTERACTION OF METHYL ORANGE WITH ELECTROLYTES
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YOSHIO~:A, K., J. Chem. Soc. Jap., 87, 324 (1966); CRESCENZI, V., QUADRIFOGLIO,F., and V. VIT.~,_GLIANO,J. Macromol. Sei., Al, 917 (1967); STRYER, L. AND BLOUT, E. R., J. Amer. Chem. Soc., 83, 1411 (1961); MoosE, J. S., PHILLIPS, G. O., PowER, D. M., AND DAVIES, J. V., J. Chem. Soc., (A), 1970, 1155, and references therein included. BRADLEY,D. F., AND WOLF, M. K., Proe. Nat. Aead. Sei. U.S. 45, 944 (1959). KLOTZ, I. M., WALKER, F. M., AND PIVAN, R. B., Y. Amer. Chem. Soc. 68, 1486 (1946). KLOTZ, I. M., J. Amer. Chem. Soe. 68, 2299 (1946). KLOTZ, I. M., AND URQUIt2~RT,J. M., J. Amer. Chem. Soe. 71, 1597 (1949). I~LOTZ,I. M., AND URQUHART,J. M., J. Amer. Chem. Soc. 71, 847 (1949). KLOTZ, I. M., BUI~KttARD, R. K., AND URQUHART, J. M., J. Amer. Chem. Soc. 74, 77 (1952). KLOTZ, I. M., AND SHIKAMA, 1~., Arch. Biochem. Biophys. 19.3, 551 (1968). KLOTZ, I. M., AND SLONIEWSKY, A. R., Biochem. Biophys. Res. Commun. 31, 421 (1968). KLOTZ, I. M., ROYER, G. P., ANn SLONIEWSKY, A. R., Biochemistry, 8, 4752 (1969). STAEDEL,W., AND BAUER, H. Ber. Bunsenges. Phys. Chem. 19, 1952 (1886). STEIN, H., AND MOORE, G. Biochem. Prep., 1, 7 (1949). R.%BINOVITCft, E., AND EPSTEIN, L. F., J. Amer. Chem. Soc. 63, 69 (1941).
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14. ZANKER, V., Z. Phys. Chem. (Leipzig) 199, 225 (1952). 15. LAMIN, M. E., AND NEVILLE, D. M., JR., J. Phys. Chem. 69, 3872 (1965). 16. BARONE, G., COSTzkNTINO, L., AND VITAGLI.A-ANO, V., Rie. Sci. 34 (II-A), 67 (1964). 17. RALSTON, A. W., AND ttOEaa, C. W., J. Amer. Chem. Soc. 64, 772 (1942). 18. HHARKINS,G. "The Physical Chemistry of Surface Films," p. 304. Reinhold, New York, 1957. 19. BOTR~, C., CRESCENZI, V., AND LIQUORI, A. M., "Original Lectures 3rd International Congress of Surface Activity," Cologne, p 302 (1960). 20. HIsI
Journal of Colloid and Interface Science, Vol. 35, No. 3, March 1971