Metachromasy: The Interactions between Dyes and Polyelectrolytes in

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19 Metachromasy: The Interactions between Dyes and Polyelectrolytes in Aqueous Solution Roger W. Kugel Department of Chemistry, Saint Mary's College of Minnesota, Winona, M N 55987

The literature on metachromasy is reviewed and two quantitative analytical methods for poly (acrylate-co-acrylamide) based on the metachromatic effect are proposed. The color-array method allows for the visual determination of polymer concentration by observing the color of toluidine blue O, cresyl violet acetate, or safranine Ο at various P/D (polymer acrylate residue to dye molecule) ratios. An abrupt color change is observed at P/D = 1. The complexation– extraction method is based on the removal of the dye Janus green Β from solution by complexation with poly (acrylate-co-acrylamide) fol lowed by extraction with 1,1,2-trichlorotrifluoroethane (Freon) sol vent. Typical concentration ranges for both tests were 0-10 ppm polymer (0-7.4 X 10 M acrylate residue) and 1-6 Χ 10 M dye. -5

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ETACHROMASY IS THE COLOR CHANGE IN THE DYE ABSORPTION SPECTRUM that occurs w h e n certain cationic dyes interact w i t h anionic polyelectrolytes i n aqueous solution. T h i s metachromatic effect was first observed i n the field o f histology w h e r e it was observed that certain dye stains changed color w h e n c o m p l e x e d b y proteins o r nucleic acids a n d could, therefore, b e used to selectively stain various subcellular structures. T h e t e r m " m e t a c h r o m a s y " was c o i n e d b y E h r l i c h to describe the appearance o f m o r e than one color i n tissue stained b y a single dye ( I ) . Since the discovery o f this p h e n o m e n o n , a large b o d y o f research has developed a r o u n d the study o f the nature o f the interaction b e t w e e n dyes a n d polyelectrolytes, the effects o f the interaction 0065-2393/93/0236-0507$07.75/0 © 1993 American Chemical Society

In Structure-Property Relations in Polymers; Urban, M., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1993.

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o n absorption o r fluorescence properties o f the dye, the types o f dyes capable o f interacting w i t h polyelectrolytes a n d u n d e r g o i n g a metachromatic shift, the types o f polyelectrolytes capable o f serving as substrates for the metachro matic dyes, a n d finally the uses to w h i c h the p h e n o m e n o n o f metachromasy can b e p u t . T h i s chapter reviews the status o f research i n the field o f metachromasy a n d proposes two n e w analytical methods f o r anionic polyelec trolytes based o n this p h e n o m e n o n .

Dyes and Polymers That Interact T h e metachromatic effect has b e e n observed between cationic dyes a n d anionic polyelectrolytes a n d b e t w e e n anionic dyes a n d cationic polyelec trolytes, although interactions o f cationic dyes have b e e n studied m o r e c o m m o n l y . Some o f the cationic dyes that interact w i t h anionic polyelec trolytes i n c l u d e acridine orange a n d its derivatives ( 2 - 7 ) , methylene b l u e (8-11), toluidine b l u e ( 1 2 , 13), brilliant cresyl b l u e (14), cresyl violet ( 1 5 ) , e t h i d i u m b r o m i d e (16), SL variety o f cyanines (12, 17, 18), a n d phthalocyanine dyes ( 1 9 ) as w e l l as a n u m b e r o f others. A n i o n i c polyelectrolytes that have the capability to i n d u c e a metachromatic color change i n cationic dyes are k n o w n as chromotropes. Polyelectrolytes that c a n serve as chromotropes for dyes i n c l u d e synthetic polyphosphates, polyacrylates, polysulfonates, polysulfates, carboxylated polysaccharides, polyphenols, a n d so forth, as w e l l as a variety o f naturally o c c u r r i n g proteins a n d nucleic acids. Actually, a relatively small fraction o f the c o m m e r c i a l l y available cationic dyes show a metachro matic shift i n the presence o f anionic polyelectrolytes. T h e dyes that d o display metachromatic shift generally d o so w i t h a w i d e variety o f polyanions, even exotic ones like 11-tungstocobaltosilicate (20). Some restrictions o n the p o l y m e r chain length o f the chromotrope w e r e discovered b y Y a m a o k a et a l . (21), w h o f o u n d a m i n i m u m critical range o f p o l y m e r chain length o f 7 - 2 0 m o n o m e r units i n polyphosphate chromotropes for metachromasy to occur. Generally, however, t h e c h e m i c a l nature o f t h e anionic sites o n the chromotrope has little effect o n the metachromatic shift exhibited b y a given dye ( 3 , 22). O n l y a f e w m i n o r polyelectrolyte-dependent differences i n metachro masy have b e e n noted. M e t h y l e n e blue, f o r example, s h o w e d an apparently greater tendency to aggregate w h e n b o u n d to p o l y (vinyl sulfate) than w h e n b o u n d to p o l y (styrene sulfonate) (11). This tendency resulted i n a greater h y p s o c h r o m i c - m e t a c h r o m a t i c shift i n the p o l y ( v i n y l sulfate) case than i n the p o l y (styrene sulfonate). H o w e v e r , the same basic absorption peaks d u e to b o u n d m o n o m e l i c (662 nm), d i m e r i e (610 n m ) , and higher aggregated forms (550 n m ) o f methylene blue w e r e observed f o r b o t h polyelectrolytes. T h e r e fore, i n this case, the different spectral changes observed for the t w o polyelectrolytes w i t h methylene b l u e were the result o f different amounts o f


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the b o u n d forms o f dye rather than inherently different c h e m i c a l interac tions.


The Nature of the Metachromatic Interaction E a r l y studies o n t h e nature o f the metachromatic interaction suggested that the spectral shifts w e r e d u e to aggregation o f the dyes b o u n d to t h e surfaces o f the polyanions (7, 19, 2 3 - 2 9 ) . S i m i l a r spectral changes w e r e observed i n solutions o f the dyes themselves at h i g h concentrations o r l o w temperatures, and these changes w e r e b e l i e v e d to b e due t o stacking o f relatively planar dye molecules ( 3 0 ) . A l t h o u g h the aggregation explanation is generally correct, the metachromatic interaction is understandably m o r e complex than simple dye aggregation. Vitagliano (31) r e v i e w e d t h e research o n metachromasy u p to 1975. Studies o f the effect o f i o n i c strength o n metachromasy f o u n d that h i g h ionic strength decreased t h e n u m b e r o f dye molecules attached to the polyelectrolyte, b u t h a d n o effect o n the spectrum o f b o u n d - d y e species ( 1 5 , 32, 3 3 ) . These observations w e r e i n t e r p r e t e d to b e the result o f the displace m e n t o f i n d i v i d u a l dye cations f r o m the p o l y m e r surface b y electrolyte cations at h i g h i o n i c strengths. H o w e v e r , because t h e b o u n d - d y e spectra i n these studies were unchanged, the simple stacking theory o f metachromasy is a n i m p e r f e c t m o d e l o f p o l y e l e c t r o l y t e - d y e structure. O t h e r studies e x a m i n e d h o w changing the dielectric constant o f the solvent affected t h e extent o f the metachromatic interaction (34, 3 5 ) . S u c h studies f o u n d that l o w e r i n g t h e dielectric constant o f the solvent decreased metachromasy. T h i s observation is understandable i n terms o f a n aggregation m o d e l because i f a dye is m o r e soluble i n a particular solvent, it w i l l show less tendency to aggregate o n a p o l y m e r surface. I n further studies, P a l a n d G h o s h (12) r e p o r t e d two types o f metachro matic interactions o f the dye pinacyanol c h l o r i d e w i t h synthetic polyanions. O n e type o f interaction caused a sharp red-shifted b a n d that was ascribed to regular stacking o f dye molecules o n the surface o f the polyanion; t h e other type o f interaction caused a b r o a d m u l t i p l e - b a n d e d spectrum that was ascribed to m o r e r a n d o m irregular interactions o f the dye molecules. R e g u l a r stacking o c c u r r e d at p o l y m e r residue-to-dye m o l a r ratios ( P / D ) o f 2, w h i c h i n d i c a t e d that w h e n dye molecules w e r e located o n alternate anionic sites o n the p o l y m e r , they w o u l d stack together i n a regular arrangement, whereas at P / D ratios near 1 the stacking was m o r e c r o w d e d a n d irregular. P a l a n d G h o s h also studied the metachromasy o f pseudoisocyanine chloride ( P I C ) i n t h e presence o f deoxyribonucleic a c i d ( D N A ) o r various v i n y l polyanion chromotropes (18). T h e y f o u n d that many, b u t n o t a l l , polyanions caused a sharp red-shifted b a n d ( k n o w n as a / band) i n t h e spectrum o f P I C . T h i s b a n d was attributed t o a particular staggered stacking


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arrangement of the P I C dye o n the alternate anionic sites o f the chromotrope that gave a staircaselike arrangement o f the angular P I C dye molecules. Shirai et al. ( 2 2 ) studied the metachromasy o f methylene b l u e w i t h poly (sodium acrylate) a n d f o u n d that the wavelength o f the metachromatic b a n d reflects the strength o f the stacking o f the b o u n d dyes: the shorter the wavelength o f the metachromatic b a n d , the stronger the stacking o f the b o u n d dyes. Scott ( 1 9 ) studied the metachromasy o f cationic phthalocyanine dyes a n d f o u n d that these dyes w e r e already aggregated i n dilute aqueous solution and, therefore, already metachromatic even before any p o l y a n i o n was a d d e d . Apparently, a metachromatic dye must be a dye that has a tendency to aggregate, but can be disaggregated i f the solution is dilute enough. I n a summary o f aggregation p h e n o m e n a i n xanthene dyes, V a l d e s A g u i l e r a a n d N e c k e r s ( 3 6 ) p r o p o s e d that parallel aggregation (stacking), w h i c h they called Η-type aggregation, resulted i n a metachromatic shift to the shorter wavelengths (hypsochromic shift) a n d linear aggregation (headto-tail), w h i c h they c a l l e d J-type aggregation, resulted i n a m e t a c h r o m i c shift to longer wavelengths (bathochromic shift). C o n d i t i o n s that favor one type o f aggregation over another c o u l d p r o v i d e structural i n f o r m a t i o n o n the p o l y m e r a n d its complexation w i t h the dye. E v i d e n c e for the importance o f the role o f electrostatic interactions i n the metachromatic effect was obtained b y C a r r o l l a n d C h e u n g (8), w h o studied the interaction o f the cationic dye methylene b l u e w i t h carboxylated starches. T h e t h e r m o d y n a m i c behavior o f the reaction i n d i c a t e d that the interaction was primarily electrostatic. T h e dye was adsorbed i n the d i m e r i c f o r m a n d i n a 1:1 ratio w i t h carboxylate groups o n the starch. G u m m o w a n d Roberts ( 3 7 ) studied the metachromasy o f anionic dyes o n the polycationic chromotrope chitosan a n d d e v e l o p e d a n e w theory for the origin o f the metachromatic effect i n general. A c c o r d i n g to this theory, metachromasy does not arise because o f adsorption o f ionic dye molecules to specific counterionic sites o n the p o l y m e r backbone, b u t rather because o f the increased concentration o f dye molecules i n the vicinity (electrostatic domain) o f the polyelectrolyte molecules. T h e aggregation o f dye molecules is t h e n a natural consequence o f this increased concentration a n d is similar to the aggregation observed at higher dye concentrations a n d driven b y hy d r o p h o b i c interactions between adjacent dye molecules i n solution. A l t h o u g h this electrostatic d o m a i n explanation is possible, it does not account for the sharp metachromatic color changes observed i n quantitative studies at P / D ratios o f 1. I n other words, the stoichiometry o f metachro masy suggests that the dye molecules coordinate to specific charged sites o n the polyelectrolyte molecule. Shirai et al. (9, 38) studied the metachromatic behavior o f methylene b l u e w i t h poly (vinyl sulfate) homologs. E v i d e n c e for saturation a n d reversal o f the metachromatic effect at h i g h P / D ratios was reported. Shirai et al. also


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observed that t h e metachromatic effect c o u l d b e reversed b y the addition o f large excesses o f K C l o r urea, a n d they f o u n d evidence that b o t h electrostatic a n d h y d r o p h o b i c interactions contribute to the b i n d i n g o f cationic dyes to polyanions. A correlation b e t w e e n the b i n d i n g strength o f dye a n d the flexibility o f the polyanion was observed. Shirai et a l . c o n c l u d e d that the stacking o f dye molecules is facilitated b y conformational changes (coiling) o f the polyanion as its charge is neutralized b y complexation o f the cationic dye molecules. T h e significance o f this w o r k for p o l y m e r studies is that metachro masy may b e used as a p r o b e o f p o l y m e r conformation. A n u m b e r o f other studies o f the nature o f t h e interaction between cationic dyes a n d anionic polyelectrolytes have b e e n carried out ( I I , 22, 39-47). F o r example, Yamagishi a n d Watanabe (40) f o u n d little dependence o n t h e alkyl c h a i n length o f N - a l k y l a t e d acridine orange interacting w i t h polystyrene sulfonic acid, Y u n et al. (41) f o u n d evidence for b o t h dye b i n d i n g a n d aggregation i n a study o f the effects o f salts a n d soluble organic c o m p o u n d s o n metachromasy, a n d Shirai et al. (22) f o u n d that t h e m a g n i tude o f the metachromatic effect was directly related to the flexibility a n d charge density o f the p o l y m e r whereas differences i n the c h e m i c a l nature o f the anionic charged sites w e r e less important. T h e p r e c e d i n g metachromasy studies and, i n particular, those o f Shirai et al. (22) a n d P a l a n d Schubert (29), suggest that t h e interaction b e t w e e n ionic dyes a n d polyelectrolytes is t h e result o f the interplay o f three types o f interactions: 1. T h e electrostatic interaction o f i o n i c dye molecules w i t h o p p o sitely charged sites o n the polyelectrolyte chain. 2. T h e h y d r o p h o b i c interaction o f dye molecules w i t h each other a n d w i t h nonpolar sites o n the polyelectrolyte. 3. T h e interaction between pi-electrons o n adjacent, adsorbed dye molecules. A m o n g these interactions, t h e t h i r d interaction probably has t h e greatest influence o n t h e spectral properties o f the adsorbed dye, because the pi-electrons o f the dye are those directly i n v o l v e d i n t h e light absorption a n d emission processes. Ionic dyes that show n o observable metachromatic effect w i t h oppositely charged polyelectrolytes c a n (and probably do) interact w i t h the p o l y m e r i c electrolytes, b u t they interact i n such a way that the pi-elec trons o f the dye are relatively unaffected b y the interaction. I n other words, i n nonmetachromatic dyes the b o u n d - d y e c h r o m o p h o r e is indistinguishable f r o m the free-dye c h r o m o p h o r e . Some evidence for this hypothesis was f o u n d i n the present study w h e r e i n certain cationic dyes that s h o w e d n o metachro matic shift w i t h poly(acrylate-co-acrylamide) i n aqueous solution still f o r m e d complexes w i t h a n d gradually coprecipitated w i t h these polymers.


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The Free-Energy Change of the Metachromatic Interaction Metachromasy occurs w h e n certain dyes a n d polyelectrolytes are present i n the same solution because the interaction lowers the free energy o f the system. Quantitative studies o f metachromasy using the p r i n c i p a l - c o m p o n e n t analysis technique w e r e c a r r i e d out ( 3 , 4 , 16, 39, 48, 49) a n d some e q u i h b r i u m constants f o r the metachromatic interaction w e r e measured. Some o f these measurements i n d i c a t e d that the entropy o f interaction p l a y e d a significant role i n the driving force for metachromasy ( 2 , 35, 42). I n other words, the entropy o f the system was significantly increased b y the interaction o f the dye w i t h the polyelectrolyte. F o r example, N i s h i d a a n d Watanabe ( 3 5 ) measured the b i n d i n g entropy o f acridine orange w i t h p o l y (sodium acrylate) i n water a n d w a t e r - o r g a n i c solvent mixtures. Values between 6 a n d 34 c a l / m o l - K f o r the entropy o f b i n d i n g , w h i c h generally increased w i t h solvent dielectric constant, were obtained. T h u s the entropy o f interaction was the greatest i n water. F u r t h e r m o r e , i n aqueous solution the b i n d i n g reaction was f o u n d to b e endothermic, so the entire driving force f o r the metachromatic interaction came f r o m the entropy t e r m . T h i s positive entropy change may b e understood i n terms o f a greater flexibility o f the polyelectrolyte chain w h e n a n u m b e r o f ionic sites are neutralized b y adsorbed dye molecules. It might also b e expected that this partial charge neutralization w i l l increase the solvation entropy.

Metachromasy and Energy Transfer Shirai et al. carried out a n u m b e r o f studies o f energy transfer i n dyes b o u n d to polyelectrolytes (11, 5 0 - 5 5 ) . T h e s e studies focused o n energy transfer i n methylene b l u e adsorbed to various polyelectrolytes, such as poly (styrene sulfonate), p o l y (vinyl sulfate), polyacrylate, a n d polymethacrylate. T h e results suggest that polyanions c a n act as templates f o r energy transfer o r c h e m i c a l reaction to b r i n g the molecular partners into close proximity f o r transfer o r reaction to occur. Aggregation o f methylene b l u e o n the polyelectrolytes t e n d e d to spoil its energy-transfer potential, presumably because o f the shorter excited-state lifetimes o f dimers a n d higher aggregates (51, 53, 55). P o l y (styrene sulfonate) a n d p o l y (vinyl sulfate) were f o u n d to b e m u c h m o r e effective than polyacrylate a n d polymethacrylate at mediating excitation e n ergy transfer between b o u n d dyes (52). T h i s mediation mechanism may b e due to the presence o f sulfur atoms i n the poly (styrene sulfonate) a n d poly (vinyl sulfate) a n d the ability o f the sulfur atoms to enhance the s i n g l e t - t r i p l e t intersystem crossing efficiency o f methylene blue. I n any event, energy-transfer rates were as m u c h as 67 times higher i n the presence o f polyelectrolytes than i n their absence. A n o t h e r interesting study o f energy transfer a n d metachromasy was carried out b y Baumgartner et a l . (56). I n this study the ability o f m e t h y l



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viologen to q u e n c h the fluorescence o f 1-naphthylamine was i n h i b i t e d b y the presence o f polyelectrolytes. I n other words, the solution phase energy transfer that n o r m a l l y occurs between 1-naphthylamine a n d m e t h y l viologen was r e d u c e d b y t h e addition o f p o l y (vinyl sulfonate) o r p o l y (styrene sul fonate) electrolytes. T h e fluorescence o f 1-naphthylamine, w h i c h is q u e n c h e d b y m e t h y l viologen, is " u n q u e n c h e d " b y further polyelectrolyte addition. I n the absence o f m e t h y l viologen, however, the polyelectrolyte h a d no effect o n 1-naphthylamine fluorescence. T h e results are interpreted i n terms o f a sequestering o f the (charged) m e t h y l viologen i n the electrostatic domains o f the polyelectrolyte. I n this case, therefore, t h e interaction between t h e dye and the polyelectrolyte actually r e d u c e d the energy-transfer potential because the polyelectrolyte d i d not concentrate t h e energy d o n o r molecule (1-naph thylamine) w h e r e t h e acceptor molecule ( m e t h y l viologen) resides. I n a classic experiment o n energy transfer between D N A a n d adsorbed acridine dyes, Isenberg et al. ( 5 7 ) observed a n d studied delayed fluorescence o f the D N A - b o u n d dyes. B a s e d o n the assumption o f an intercalation m o d e l o f adsorption [the relatively flat acridine dyes slide between t h e base pairs o f D N A ( 5 8 , 59)], they c o n c l u d e d that w h e n a D N A c h r o m o p h o r e (base pair) absorbs light, some o f the energy o f the excited singlet state is converted into the lower energy triplet state, w h i c h is a l o n g - l i v e d state. T h e triplet energy can j u m p f r o m base pair to base pair u p a n d d o w n the chain d u r i n g its l o n g lifetime u n t i l i t decays spontaneously b y D N A phosphorescence, degrades to heat, or, i f dye is present, transfers to a n intercalated d y e molecule a n d triggers delayed fluorescence. T h i s observation o f triplet migration, some times called exciton transfer o r triplet d e r e a l i z a t i o n ( 6 0 ) , also substantiated the intercalation m o d e l for acridine dye adsorption to D N A because tripletto-singlet energy transfers f r o m D N A to dye must take place over very short (contact) distances.

Metachromasy as a Structural Probe A n u m b e r o f studies have examined the effects o f structural properties o f polyelectrolytes o n metachromasy. Because i n many instances the metachro matic effect is sensitive to structural changes, it may b e u s e d as a structural p r o b e for polyelectrolytes. T y p i c a l p o l y m e r structural properties measured b y metachromatic techniques i n c l u d e intrinsic viscosity, conformation, a n d chain length, but other properties may b e p r o b e d as w e l l . T h e studies covered here i n c l u d e both absorption and fluorescence techniques, a n d these topics w i l l b e used as a convenient, though somewhat arbitrary, division f o r the review.

Metachromatic Absorption Studies of Polymer Structure. A b s o r p t i o n studies i n c l u d e those metachromatic experiments i n w h i c h U V - v i s i b l e absorption spectrophotometry is t h e p r i m a r y t o o l f o r analysis. O t h e r physical measurement techniques, such as dialysis o r viseosimetry, are



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often u s e d i n conjunction w i t h the spectrophotometry but do not constitute the p r i m a r y m e t h o d o f use. Structural parameters o f the p o l y e l e c t r o l y t e s — conformation, c h a i n length, charge density, hydrophobicity, tacticity, distance o f charged sites f r o m p o l y m e r backbone, e t c . — m a y be v a r i e d systematically a n d the effects o n the metachromasy o f various dyes noted. T h o s e parameters that have a consistent, predictable effect o n the metachromasy o f a particular dye may be p r o b e d b y that dye i n an u n k n o w n p o l y m e r system. C a u t i o n must be used i n interpretation o f the results o f such experiments, however, because changes i n metachromasy are fairly nonspecific. I n other words, a variety o f changes i n structural parameters or even conditions o f the experi ment may cause similar metachromatic changes w i t h a given dye. C a r e must be taken, therefore, to ensure that o n l y one parameter has b e e n v a r i e d before metachromatic changes are assigned to changes i n that parameter. Vitagliano (31) reported the effect o f p o l y m e r conformation o n the metachromasy o f acridine orange. I n this study, acridine orange was b o u n d to polyacrylic acid a n d polymethacrylic a c i d at P / D ratios o f 7600 a n d 8000 u n d e r acidic conditions at l o w degree o f neutralization ( a ) values. U n d e r these conditions the polyelectrolytes were assumed to be i n a tightly c o i l e d f o r m because o f their hydrophobicity, a n d the acridine orange showed the characteristic spectrum o f m o n o m e l i c b i n d i n g to the polyelectrolytes. A s base was a d d e d a n d a increased, the polymers underwent a conformational change (at a — 0.1-0.3) as they became m o r e i o n i z e d a n d the acridine orange spectrum shifted to the characteristic spectrum o f aggregate b i n d i n g . I n other words, i n this system the adsorbed acridine molecules c o u l d migrate a n d aggregate m o r e readily w h e n the polyelectrolytes t o w h i c h they were b o u n d were i n an o p e n rather than c o i l e d f o r m . O t h e r things b e i n g equal, u n d e r these conditions acridine orange c o u l d b e used as a sensitive p r o b e o f polyacrylic acid or polymethacrylic a c i d conformation. N

N

N

Shirai et al. also studied the effects o f conformational changes o f p o l y electrolytes o n metachromasy (9, 22, 38, 61). I n these studies the interaction o f methylene b l u e w i t h various polyelectrolytes was examined spectrally. T h e results suggested that important factors for metachromasy are the flexibility a n d charge density o f the p o l y m e r : the more flexible the polyanion o r the higher its charge density, the greater was the tendency o f adsorbed methy lene b l u e t o aggregate. Viscosity measurements (38) also indicated that conformational changes i n the polymers c o u l d b e i n d u c e d b y dye adsorption; that is, b i n d i n g o f cationic dyes t o anionic polyelectrolytes neutralizes the charge o f the p o l y a n i o n a n d causes the p o l y m e r molecules t o c o i l u p . M e t h y l e n e b l u e was f o u n d t o aggregate m o r e readily w h e n adsorbed t o polymers i n the f o l l o w i n g order (22): poly (sodium maleate-co-vinyl alcohol) > p o l y (sodium acrylate) > p o l y (sodium methaerylate). T h e poly (sodium maleate-co-vinyl alcohol) might b i n d more effectively t o the cationic dye because o f its v i c i n a l anionic sites, a n d the p o l y (sodium acrylate) might allow dye aggregation m o r e effectively than the p o l y (sodium methaerylate) because


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o f its higher flexibihty. T h u s , t h e ability o f a polyanion to i n d u c e metachro masy i n methylene b l u e might b e used as a probe f o r flexibihty o r charge density o f the polyanion. I n a further study o f p o l y m e r conformation G u h a n i y o g i a n d M a n d a i (10) observed the metachromatic effect o f the dye pinacyanol w h e n it interacted w i t h short-chain benzene-soluble polymers that h a d carboxylate e n d groups. I n this case, the dye showed a metachromatic shift due to d i m e r i z a t i o n u n d e r conditions that allowed the intramolecular association o f p o l y m e r e n d groups. Therefore, the observed spectral shift was used as a probe o f p o l y m e r conformation i n nonaqueous m e d i a . I n some studies the degree o f p o l y m e r i z a t i o n o f the polyanion also was f o u n d to affect the ability o f a dye to engage i n a metachromatic interaction w i t h t h e polyanion ( 9 , 21, 38, 62). F o r example, Y a m a o k a et al. (21) studied the metachromatic interaction o f crystal violet w i t h s o d i u m polyphosphates o f varying chain length. T h e metachromasy o f this dye was f o u n d to increase sharply i n a narrow range o f chain lengths f r o m 7 t o 20 m o n o m e r phosphate units. A n effect o f degree o f polymerization o n metachromasy also was seen to a certain extent w i t h carbon-backbone polyanions ( 9 ) . H o w e v e r , above a critical m i n i m u m value, t h e degree o f polymerization h a d a n insignificant effect o n the strength o f the metachromatic interaction (22). T h e effects o f p o l y m e r c h a i n tacticity were also studied using metachro masy (22, 31, 63). I n a study o f the interaction o f acridine orange w i t h various polyelectrolytes, f o r example, i t was f o u n d that t h e b i n d i n g strength was higher a n d the stacking coefficient was l o w e r w i t h isotactic poly methacrylic a c i d a n d poly(styrenesulfonic acid) than w i t h the corresponding atactic polyacids (63). T h e results were interpreted i n terms o f a relatively stable helical conformation o f the isotactic polyacids w i t h the carboxylate groups facing outward. T h e dye c a n associate readily w i t h these exposed anionic groups, b u t the helical conformation might b e m o r e difficult to disrupt to facilitate dye aggregation. It has also b e e n suggested (31) that i n the case o f isotactic p o l y (styrene sulfonate) the arrangement o f aromatic groups a r o u n d t h e helical backbone c h a i n may allow some k i n d o f partial intercalation o f dye molecules similar to that p r o p o s e d for the interaction o f acridine dyes w i t h D N A (64). F u r a n o et a l . ( 6 5 ) described the use o f the metachromasy o f acridine orange to p r o b e the conformation o f ribonucleic a c i d ( R N A ) i n ribosomes. C o m p a r i s o n o f the sensitivity o f the extinction coefficient o f acridine orange w i t h t h e P / D ratio a n d t h e dye stacking coefficient Κ f o r a variety o f o r d e r e d and disordered R N A s y i e l d e d a useful correlation. I n particular, a highly o r d e r e d (double-helical) R N A gave a r a p i d loss i n extinction o f acridine dye w i t h p o l y m e r addition a n d a l o w stacking coefficient, whereas a disordered R N A gave a m o r e gradual loss i n extinction a n d a h i g h stacking coefficient. T h i s test was a p p l i e d to ribosomal R N A a n d l e d to the conclusion that R N A i n the ribosomes h a d little double-helical structure. T h i s c o n c l u -


516



sion, i n t u r n , resulted i n a general m o d e l o f ribosomal structure. S i m i l a r results b y Stone a n d Bradley established acridine orange as a structural p r o b e for D N A conformation (66). R e c e n t l y P a l a n d M a n d a i (98) used the metachromatic interactions o f 1,9-dimethyl methylene b l u e a n d pinacyanol w i t h potassium alginate poly mers to probe conformational changes of these polymers. C i r c u l a r d i c h r o i s m i n d u c e d i n the spectra o f these dyes suggested that the dye molecules c o m p l e x e d w i t h the polymers i n a helical arrangement. T h e s e observations p r o m p t e d P a l a n d M a n d a i to infer a left-handed helical conformation for potassium alginate i n solution. G e n e r a l results o f such experiments must be interpreted w i t h caution, however, because h i g h concentrations o f dyes aggregated o n a p o l y m e r conceivably c o u l d influence the conformation o f the polymer.

Metachromatic Fluorescence Studies of Polymer Structure. L u m i n e s c e n c e techniques i n general have b e e n used w i d e l y a n d successfully i n the study o f polymers and their properties. M a n y of these studies involve polymers that have covalently attached fluorescent groups or pendant side chains. Because metachromasy technically involves only the interaction be tween polymers a n d dyes that are not covalently attached, this chapter w i l l not address structural studies o f inherently fluorescent polymers or polymers covalently tagged w i t h fluorescent probes. T h e reader is referred, instead, to excellent reviews o n this subject (67-70). Fluorescence metachromasy as a p o l y m e r structural probe may be d i v i d e d into two categories: interactions w i t h naturally o c c u r r i n g polymers a n d interactions w i t h synthetic polymers. Naturally Occurring Polymers. Fluorescence metachromasia cov e r e d i n an early review b y Stockinger (71) also h a d its origins i n the field o f biological stain technology. C e r t a i n dyes, k n o w n as fluorochromes, b i n d to specific naturally o c c u r r i n g polymers or complexes and highlight subcellular structures where these p o l y m e r compounds are concentrated. T h i s highlight i n g allows the subcellular structures to be v i e w e d b y fluorescence microscopy. A c r i d i n e orange presents a classic example o f the usefulness o f this m e t h o d because, u n d e r the p r o p e r conditions, acridine orange c o m p l e x e d w i t h D N A fluoresces yellow-green, whereas acridine orange complexed w i t h R N A fluo resces red-orange. Therefore, the chromosomes i n a cell nucleus ( D N A ) can be distinguished readily f r o m the nucleolus ( R N A ) b y u s i n g a single stain procedure. This dual fluorescence arises because o f differences i n the inter actions between acridine a n d the two nucleic acids, a n d can be used as a probe for D N A or R N A . O t h e r dyes serve as effective fluorochromes for proteins, carbohydrates, a n d lipids (72) i n the fluorescent staining o f biologi cal structures. Some o f the uses o f these fluorescing molecules for the elucidation o f naturally o c c u r r i n g p o l y m e r structures are s u m m a r i z e d i n the following paragraphs.



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P a l (24) studied the fluorescence o f acridine orange a n d rhodamine 6 G i n the presence o f natural polyelectrolytes like D N A , c h o n d r o i t i n sulfate, a n d λ-carageenan. A b s o r p t i o n metachromasy a n d fluorescence q u e n c h i n g were observed to different extents for b o t h dyes, a n d the effects were f o u n d to b e reversable b y the addition o f salts, urea, o r alcohols. T h e s e results were not surprising because b o t h effects (metachromasy a n d quenching) were k n o w n to accompany dye aggregation at higher concentrations. N i l e r e d is a fluorescent dye that has an emission wavelength that is a sensitive function o f solvent polarity. I n hydrocarbons, N i l e r e d fluoresces yellow-gold whereas i n ethanol it fluoresces r e d (73). T h i s fact was u t i l i z e d i n a n o v e l w a y i n a study i n w h i c h N i l e r e d was used as a polarity-sensitive fluorescent probe o f h y d r o p h o b i c p r o t e i n surfaces (74). Interaction o f N i l e r e d w i t h various types o f proteins enhanced a n d blue-shifted the fluorescence o f the dye to a n extent related to interaction o f the dye w i t h the h y d r o p h o b i c domains o n the p r o t e i n surfaces. T h e dye c o u l d b e used to m o n i t o r partial denaturation o f a protein a n d the concomitant exposure o f h y d r o p h o b i c groups b y following the progressive shift i n fluorescence wavelength a n d enhancement o f fluorescence intensity. T h i s use o f N i l e r e d as a denaturation probe was demonstrated w i t h the p r o t e i n o v a l b u m i n . A n u m b e r o f other studies have focused o n the use o f fluorescent probes to study proteins i n solution ( 7 5 - 7 7 ) . F o r example, l-anilino-8-naphthalene sulfonate, dansylamide, a n d other fluorophores have b e e n used to character ize a n d quantitate d r u g b o n d i n g sites o n serum a l b u m i n b y various c o m p e t i tion studies (78-80). I n addition, auramine Ο was used as a fluorescent probe f o r t h e quantitation a n d d r u g b i n d i n g site analysis o f α-acid glycopro tein (81). S u c h studies show the usefulness o f certain fluorescent dyes i n characterizing particular proteins i n c l u d i n g specific b i n d i n g site analyses. Synthetic Polymers. L e v s h i n et a l . (82) studied the interaction o f rhodamine 6 G ( R h 6 G ) w i t h various synthetic sulfonate polymers. O b s e r v e d interactions between the dye a n d polyelectrolytes were accompanied b y typical metachromatic absorbance changes i n the dye spectrum as w e l l as fluorescence quenching, b o t h o f w h i c h reversed at h i g h P / D ratios. T h e effects were m o r e dramatic w i t h poly (styrene sulfonate) than w i t h p o l y (vinyl sulfonate), probably o w i n g to the enhanced h y d r o p h o b i c interaction between the dye a n d the aromatic side chain groups i n the p o l y (styrene sulfonate). Additionally, a higher charge density o n the polyelectrolyte magnified the fluorescence q u e n c h i n g effect that a p o l y m e r exerted o n R h 6 G . F e n y o et al. (83) as w e l l as B r a u d (84) used the interaction o f auramine Ο w i t h various synthetic polycarboxylic acids to probe the structural parame ters o f the p o l y m e r . I n their experiments, auramine Ο was f o u n d to b e an effective and sensitive probe to establish the presence o f h y d r o p h o b i c regions i n synthetic polymers or, at least, the existence o f special compact conforma tions. A u r a m i n e Ο fluoresced intensely w h e n it was p l a c e d i n contact w i t h


518


such regions o n a tightly c o i l e d n o n i o n i z e d synthetic polyacid. T h e fluores cence disappeared, however, w h e n the p o l y m e r unravelled or became i o n i z e d . T h u s , i n such p o l y m e r systems, the fluorescence o f auramine Ο can be used as a direct measure o f p o l y m e r conformation or degree o f ionization.


Similar behavior was observed w i t h crystal violet (47). T h i s dye fluo resces strongly i n interaction w i t h undissociated polymethacrylic acid, but only weakly fluoresces w h e n the p o l y m e r dissociates 4 0 % o r more. T h u s crystal violet may also serve as a conformation-charge-density probe for polymethacrylic acid. M u l l e r a n d F e n y o (85) studied the fluorescence metachromasy o f acri dine orange i n the presence o f polyacryhc acid, polymethacrylic acid, a n d a polycondensate between 1,3-benzenedisulfonyl chloride a n d L-lysine. T h e y f o u n d that acridine orange c o u l d b e used as a sensitive p r o b e o f p o l y m e r conformation, especially i n the case o f the polycondensate. W h e n the dye binds to a p o l y m e r that is i n the c o i l e d undissociated state, it binds i n the m o n o m e l i c f o r m a n d its green fluorescence (535 n m ) is enhanced. A s the p o l y m e r unfolds w i t h a c i d group dissociation, the green fluorescence disap pears a n d is replaced b y the characteristic r e d fluorescence (640 n m ) o f acrindine dimers a n d higher aggregates. T h e conformational state o f the p o l y m e r can be assessed b y the color o f acridine orange fluorescence; hence, this dye may be used as a visual p r o b e o f p o l y i o n conformation. I n another study o f p o l y m e r conformation b y metachromasy, e t h i d i u m b r o m i d e a n d auramine Ο were f o u n d to be useful as fluorescent metachro matic probes o f the conformational states o f maleic a c i d - o l e f i n copolymers (86). I n this study, metachromatic results were correlated w i t h viscosity a n d e q u i h b r i u m constant measurements to verify the conformational states o f the polymers, a n d the fluorescence o f e t h i d i u m b r o m i d e was f o u n d to be sensitive to the structure of the c o i l e d polymer. Possible similarities between the behavior o f e t h i d i u m b r o m i d e i n this system a n d its intercalation behavior w i t h D N A were suggested. A n o t h e r study examined the interaction i n aqueous solution o f pyrene substituted w i t h a positive side group a n d a polyanion as w e l l as pyrene substituted w i t h a negative side group a n d a polycation (44). I n either case, the metachromatic results were the same: T h e pyrene absorption spectrum b r o a d e n e d a n d its p r i n c i p l e fluorescence bands shifted f r o m 377 a n d 400 n m to a b r o a d b a n d centered a r o u n d 480 n m . T h e 4 8 0 - n m fluorescence b a n d was believed to be due to excimer fluorescence f r o m pyrene dimers and higher aggregates. T h u s , the fluorescence shift o n contact w i t h polyelectrolytes was taken as evidence for aggregation o f p o l y m e r - b o u n d pyrene moieties a n d c o u l d be used as yet another p r o b e o f polyelectrolyte conformation. M e t a c h r o m a s y is useful as a structural probe for polyelectrolytes. T h e effect relies o n the fact that spectral properties change w h e n certain dyes aggregate or f o r m clusters. T h e appearance o f d i m e r absorption bands or excimer fluorescence bands signals the onset o f dye molecule aggregation,


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w h i c h is facilitated b y polyelectrolytes whose structural properties affect the strength o f the p o l y m e r - d y e interaction. T h e ability o f a given dye to i n d u c e metachromasy is affected w h e n a p o l y m e r changes structure. T h u s metachro matic color changes are sensitive to p o l y m e r structural changes a n d m a y b e used as effective structural probes.


Metachromasy as an Analytical Tool It was observed very early i n t h e history o f metachromasy that the stoichiometry o f the m a i n interaction between a charged dye a n d a n oppositely charged polyelectrolyte is 1:1 (8, 12, 13). That is, metachromasy, i f i t occurs, usually increases to a m a x i m u m as the P / D (polymer charged residue to dye molar concentration) ratio increases f r o m 0 to 1. A t a P / D ratio o f 1 the metachro matic effect typically levels off, a n d at very h i g h P / D values i t reverses ( 7 ) . I f the metachromatic reaction is " c l e a n " a n d stoichiometric (i.e., i f the dye a n d polyelectrolyte react quickly, f o r m a strong complex, a n d leave very little free dye i n solution at t h e equivalence point), a n d i f t h e c o m p l e x e d dye c o m pletely aggregates at P / D ratios less than 1, t h e n the process may b e u s e d to quantify the concentrations o f dye o r p o l y m e r i n the P / D range f r o m 0 to 1. Some examples o f such quantitative analyses are given i n the following text. G o r m a l l y et a l . ( 9 0 ) developed a m e t h o d that used the interaction between toluidine b l u e a n d carboxymethyl cellulose to measure t h e dye concentration over a w i d e range o f p o l y m e r a n d dye values w i t h good reliability. Some inherent advantages o f this m e t h o d over direct spectrophotometrie o r fluorometric techniques were discussed. M e t a c h r o m a t i c methods have b e e n developed f o r the quantitative deter mination o f many naturally o c c u r r i n g polyelectrolytes, such as arylsulfatase (91), carageenan (92), glycosaminoglycans (46), h e p a r i n (93), a n d others. A l t h o u g h these methods typically involve standard absorptive metachromatic shifts, W u (94) described a n interesting secondary m e t h o d involving fluores cence metachromasy: T h e fluorescence intensity o f certain polycyclic aro matic dyes significantly increased w h e n the dyes interacted w i t h various cationic o r n o n i o n i c polymers. T h e s e complexes also h a d a n affinity f o r certain naturally o c c u r r i n g biological polyanions, a n d t h e resulting ternary complexes c o u l d b e used i n various quantitative methods, such as fluores cence microscopy or flow cytometry. I n this m e t h o d the intermediate cationic or nonionic p o l y m e r served as a mordant to fix the fluorescent dye to the naturally o c c u r r i n g analyte. O p t i c a l o r spectrophotometric titration has b e e n used to quantify the metachromatic interaction (2, 63). I n this m e t h o d the absorbance o f the m a i n visible b a n d o f the free dye i n solution was m o n i t o r e d as a f u n c t i o n o f p o l y m e r (titrant) concentration o r Ρ / D ratio. A n example o f such a titration curve is shown i n F i g u r e 1. T h e titration curves f o r clean metachromatic



520


1

2

3

4

5

p/D

Figure 1. Spectrophotometric titration of acridine orange with isotactic poly (styrene sulfonate); dye concentration 10 ~ M. The end point corresponds to the 1:1 ratio between dye concentration and polyelectrolyte equivalent {S0 _ groups) concentration. (Reproduced with permission from reference 63. Copyright 1976.) 5

3

reactions are characterized b y a linear decrease i n absorbance i n the P / D range 0 - 1 f o l l o w e d (ideally) b y a n abrupt change i n slope w i t h n o further decrease i n absorbance. T h e e n d p o i n t o f such a titration is d e t e r m i n e d as t h e intersection o f the t w o linear segments o f the curve a n d typically occurs at P / D = 1.0. N o t e that the titration curve s h o w n i n F i g u r e 1 was measured u n d e r i d e a l conditions: acridine orange interacted w i t h isotactic poly (styrene sulfonate), a metachromatic reaction that is especially " c l e a n " . T i t r a t i o n curves measured u n d e r less o p t i m u m conditions (with weaker metachromatic dyes o r less o r d e r e d o r nonaromatie polyelectrolytes) w i l l typically show nonlinearity i n b o t h segments o f the curve o r less abrupt e n d points. F u r t h e r m o r e , very often the metachromatic reaction is not instantaneous; spectral changes c a n take place over 30 m i n to 1 h ( C . Pierce, N a l c o C h e m i c a l C o m p a n y , u n p u b l i s h e d results), w h i c h makes exact quantitation very difficult. I n addition, the metachromatic reaction is often accompanied


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b y partial coagulation o f the d y e - p o l y e l e c t r o l y t e complex that c a n raise t h e turbidity o f the solution a n d further complicate spectral measurements. D e s p i t e these complications, metachromasy has b e e n p r o p o s e d as a m e t h o d o f quantitation o f polycarboxylate o f polysulfonate concentrations d o w n to the 1 - p p m level ( 9 5 ) using pinacyanol o r N i l e b l u e A as the metachromatic dye. T h i s m e t h o d f o u n d reasonable linearity i n the metachro matic calibration curves over narrow ranges o f dye concentration. F u r t h e r more, the m e t h o d c o u l d analyze separately f o r polycarboxylates a n d polysulfonates i n the same sample b y adjusting the p H o f the system. A n o t h e r m e t h o d o f quantitation for cationic polyelectrolytes was r e p o r t e d b y Parazak et a l . ( 9 6 ) . T h i s m e t h o d utilized the interaction between anionic dyes a n d cationic polyelectrolytes. H o w e v e r , instead o f r e q u i r i n g a metachro matic spectral change u p o n interaction, the m e t h o d utilizes t h e fact that the neutral complex, w h i c h is somewhat water-insoluble, m a y b e extracted f r o m aqueous solution b y a h y d r o p h o b i c solvent. I n this m e t h o d a 1,1,2-trichlorotrifluoroethane ( T C T F E ; F r e o n ) solvent was used, a n d t h e neutral complex was attracted to the T C T F E - w a t e r interface w h e r e it f o r m e d a w e b l i k e structure. T h e d y e that r e m a i n e d i n aqueous solution was t h e n measured spectrophotometrically a n d the loss i n absorbance was f o u n d to b e directly proportional to t h e concentration o f polyelectrolyte. T h i s m e t h o d was f o u n d to b e sensitive d o w n to 0.5 p p m o f cationic polyelectrolyte. Finally, O n a b e ( 9 7 ) observed that the visible metachromatic color change o f toluidine blue f r o m light b l u e i n the free cationic state to r e d - p u r p l e i n reaction w i t h anionic polyelectrolytes was abrupt a n d dramatic. T h i s observa t i o n l e d to t h e suggestion that this color change c o u l d b e used to visually detect the e n d p o i n t i n a c o l l o i d titration; that is, toluidine b l u e c o u l d serve as a c o l l o i d titration indicator.

Color-Array and Complexation-Extraction Studies Because the metachromatic spectral change occurs linearly over a narrow range o f P / D values a n d because the metachromatic organic dyes generally have high molar absorptivities, the metachromatic effect has b e e n p r o p o s e d as a basis f o r t h e quantitative analysis o f synthetic polyelectrolytes i n the 1 - 1 0 - p p m range i n water ( 9 5 , 9 6 ) . A l t h o u g h the technique does w o r k , its sensitivity to temperature, p H , i o n i c strength, a n d saturation make applica t i o n difficult, especially i n the field. A n e w set o f standards must b e created for every n e w water sample a n d sometimes reproducibility is difficult to attain o n separate days even w i t h the same water sample. F u r t h e r m o r e , a time dependence f o r aggregation has b e e n observed i n some instances w h e r e i n the metachromatic spectral change does not occur instantaneously, b u t develops gradually over several minutes o r hours. I n addition, over l o n g t i m e periods a gradual settling out o f the " d y e d " neutral polyelectrolyte



522


occasionally has b e e n observed. T h e aforementioned effects certainly c o m p l i cate the use o f metachromasy for p o l y m e r analysis. I n o u r study, the use o f the metachromatic effect for low-level polyelec trolyte analysis was reexplored w i t h a n e w twist. Because the metachromatic color change takes place over a n a r r o w fixed P / D range, w e v a r i e d the dye concentration at a fixed p o l y m e r concentration a n d visually observed the dye concentration that caused the spectral shift to take place. T h i s process was repeated at several standard p o l y m e r concentrations to generate a standard color array (i.e., color o f solution versus p o l y m e r a n d color o f solution versus dye concentration) against w h i c h a set o f solutions w i t h an u n k n o w n p o l y m e r concentration a n d various k n o w n dye concentrations c o u l d be visually c o m pared. Some cationic metachromatic dyes have spectral changes u p o n c o m plexation w i t h s o d i u m p o l y (acrylate-co-acrylamide) that are significant e n o u g h to be readily detectable visually. T h e behavior o f these dyes was examined i n o u r study. I n addition, w e f o u n d that the m e t h o d o f Parazak et al. (96) m a y be a p p l i e d to anionic polyelectrolytes such as s o d i u m p o l y (acrylate-co-acrylam i d e ) . W h e n certain cationic dyes reacted w i t h this p o l y m e r , the resultant complex c o u l d b e extracted into a T C T F E - w a t e r interface. T h i s extraction process r e m o v e d dye a n d polyelectrolyte f r o m the aqueous phase i n constant p r o p o r t i o n . T h u s the loss i n dye absorbance after complexation a n d extraction was p r o p o r t i o n a l to the initial concentration o f polyelectrolyte. T h e p o l y m e r concentration c o u l d be assessed either visually o r spectrophotometrically b y this complexation-extraction p r o c e d u r e .

Experimental Details D y e s a n d extraction solvents used i n this study w e r e obtained f r o m A l d r i c h C h e m i c a l C o m p a n y a n d w e r e u s e d without further purification. Solutions w e r e b u f f e r e d to p H 7.0 using a phosphate buffer. S o d i u m poly (acrylate-coacrylamide) samples w e r e obtained f r o m N a l c o C h e m i c a l C o m p a n y . U V - v i s i b l e spectra w e r e r u n o n a spectrophotomer ( I B M 9430). A c o p o l y m e r o f 7 0 % acrylic acid a n d 3 0 % acrylamide w i t h a molecular weight range o f 2 5 , 0 0 0 - 4 0 , 0 0 0 g / m o l was used.

Results F r o m 39 cationic dyes screened for their ability to manifest a visual metachromatic effect, 3 dyes w e r e chosen: toluidine b l u e O , cresyl violet acetate, a n d safranine O . T h e structures o f these dyes are s h o w n i n C h a r t I. I n this test, an array o f solutions was made u p w i t h varying p o l y m e r a n d dye concentrations. T h e p o l y m e r concentrations w e r e 0, 2.5, 5.0, 7.5, a n d 10.0 p p m a n d the dye concentrations w e r e 1, 2, 3, 4, 5, a n d 6 X 1 0 " M . T h u s , 5


KUGEL

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(a) toluidine blue 0

CH C0 3

12

U

"

2

IT^

(b) cresyl violet acetate CH NH

9

cr

(c) safranine 0

(CHCH) Ν 3

2 2

cr ^N(CH)

32

(d) janus green Β Chart I. Dyes used for color-array and complexation-extraction studies.


524


for each dye an array o f thirty 1 0 0 - m L solutions was p r e p a r e d a n d p h o tographed. C o p i e s o f these photographs are shown i n Plates 1 - 3 . I n these color-array tests, the p o l y m e r concentration that caused a metachromatic shift i n the dye increased w i t h increasing dye concentration. W h e n the molar concentrations o f carboxylate residues i n the p o l y m e r solutions were deter m i n e d a n d d i v i d e d b y the molar dye concentrations, the resulting P / D ratios were calculated. A chart of P / D ratios for each solution i n the array was made a n d is presented i n F i g u r e 2. A comparison o f the Ρ / D values o n this Downloaded by NORTH CAROLINA STATE UNIV on June 22, 2013 | http://pubs.acs.org Publication Date: May 5, 1993 | doi: 10.1021/ba-1993-0236.ch019

chart a n d the colors i n the t w o - d i m e n s i o n a l arrays shows that the significant metachromatic color change occurs over a fairly narrow P / D ratio range near unity. T h i s is consistent

with

previous w o r k that

established

that

the

metachromatic effect was the strongest at P / D values o f 1.0 (13). T h e color-array results for t o l u i d i n e b l u e Ο give the most dramatic visual metachromatic

effect, the results for cresyl acetate also show a readily

discernible color change, a n d the results for safranine Ο indicate very little effect o n the photograph. ( N o t e that color changes i n the safranine Ο array w e r e subtle but noticeable visually w h e n the solutions w e r e photographed. Unfortunately these color changes d i d not c o m e through i n the pictures.) T h e peak absorbance values for solutions i n each o f the color-array tests are shown i n Figures 3 - 5 . T h e s e graphs indicate a general decrease i n dye absorbance as p o l y m e r is a d d e d t h r o u g h P / D = 1. Some o f the metachromatic solutions w e r e extracted w i t h nonaqueous solvents, such as toluene, methylene chloride, or T C T F E . T h e nonaqueous solvent r e m o v e d the d y e - p o l y m e r complex f r o m aqueous solution a n d the resulting aggregate was attracted to the interface between the two liquids, w h e r e it f o r m e d a w e b l i k e film. T h i s c o l o r e d film was readily seen w h e n the two-phase mixture was shaken, but the

film

nearly disappeared i n the

interface w h e n the mixture stood u n d i s t u r b e d . T h e p h e n o m e n o n accentuated the differences b e t w e e n equivalent cationic dye solutions w i t h a n d w i t h o u t 1.0 p p m o f polyanion, a n d c o u l d be used to determine the concentration o f p o l y m e r i n solution m a i n l y b y depletion o f the dye color i n the phase.

aqueous

S u c h a m e t h o d was p r o p o s e d for anionic dyes c o m p l e x i n g w i t h

polycations ( 9 6 ) a n d was f o u n d to be sensitive d o w n to 0.5 p p m o f polyelec trolyte. T h e extraction results o b t a i n e d i n this study suggest the feasibility o f application o f this technique to the analysis o f l o w levels o f polyanions. Solutions that contain the dye Janus green Β a n d s o d i u m poly(acrylate-coaerylamide) at P / D ratios near 1 w e r e successfully extracted w i t h 1,1,2-trichlorotrifluoroethane ( F r e o n ) a n d the aqueous layer was left nearly colorless. Plate 4 is a photograph of the solutions that resulted f r o m an extraction experiment i n w h i c h 1 X 10 ~

5

M Janus green Β was m i x e d w i t h 0, 1, a n d 5

p p m o f s o d i u m poly(acrylate-co-acrylamide). A s the figure clearly indicates, the complexation-extraction m e t h o d is visually sensitive d o w n to a p o l y m e r


19.

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Metachromasy

527

10.0

7.4

3.7

2.5

1.8

1.5

1.2

7.5

5.6

2.8

1.8

1.4

1.1

0.9

5.0

3.7

1.8

1.2

0.9

0.7

0.6

2.5

1.8

0.9

0.6

0.5

0.4

0.3

0

0

0

0

0

0

0

1

2

3

4

5

6

ε QL

a. c

ο

Polymer Concent


V> -

Dye Concentration

(10-5M)

Figure 2. Chart of P/D ratio values for each of the solutions in the color-array studies shown in Plates IS. concentration o f 1 p p m o r less. T h e structure o f the dye Janus green Β is shown i n C h a r t I.

Discussion T h e experimental results suggest that f o r dyes that exhibit a metachromatic shift w i t h poly(acrylate-co-acrylamide), the color change occurs over a n a r r o w P / D range near unity. T h e P / D ratio represents t h e n u m b e r o f anionic p o l y m e r residues p e r dye molecule i n the system. I n this copolymer, 3 0 % o f the residues are n o n i o n i c acrylamide, w h i c h is apparently metachromatically inactive. T h e observation o f color change at P / D = 1 is consistent w i t h many previous results that established P / D ratios near 1 f o r the m a x i m u m metachromatic effect. T h i s stoichiometry also emphasizes t h e importance o f the electrostatic interaction b e t w e e n t h e cationic dye a n d t h e anionic poly electrolyte residue vis â vis the h y d r o p h o b i c a n d pi-electron interactions. Because metachromatic change occurs over a small range o f P / D , i t is difficult to use metachromasy f o r the linear analysis o f p o l y m e r solutions. T h e results i n t h e literature also suggest that as P / D increases b e y o n d 1, t h e




528

metachromasy saturates very quickly a n d begins to reverse at P / D greater than 10 ( 7 ) . D e p e n d e n t o n the dye concentration, the linear useful range o f spectral change versus p o l y m e r concentration is often too narrow to be used. I n o u r study, the narrowness o f the metachromatic range was used to advantage. W h e n p o l y m e r solutions were m i x e d w i t h several concentrations



19.

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529

o f the same metachromatic dye, the solutions w i t h p o l y m e r (anionic residue) concentration less than the dye concentration d i d not show a color change, whereas the solutions w i t h p o l y m e r (anionic residue) concentration greater than the dye concentration d i d change color. I n other words, color change o c c u r r e d at P / D = 1 w i t h the metachromatic color manifest at P / D > 1 a n d the free dye color seen at P / D < 1. Because the dye concentrations i n these test solutions were k n o w n , the p o l y m e r (anionic residue) concentration c o u l d readily be d e t e r m i n e d . F u r t h e r m o r e , t o l u i d i n e b l u e Ο a n d cresyl violet acetate gave p r o n o u n c e d visual color changes so that the analytical test c o u l d be carried out without a spectrophotometer or colorimeter, a n d a simple color w h e e l or chart c o u l d be used to determine p o l y m e r concentrations. T h e p r e l i m i n a r y extraction studies i n w h i c h solutions that contained cationic dye a n d anionic polyelectrolyte w e r e extracted w i t h a nonaqueous solvent like T C T F E may also b e a p p l i e d to p o l y m e r analysis. Because the neutral d y e - p o l y m e r complex migrated to the interface between the two i m m i s c i b l e liquids a n d was effectively r e m o v e d f r o m the aqueous layer, any r e m a i n i n g color i n the water layer must be attributable to excess dye (presuming a strong interaction between dye a n d p o l y m e r so that complexat i o n was complete). T h e p o l y m e r concentration i n such systems can be d e t e r m i n e d spectrophotometrically (by analysis o f the residual dye i n the water layer using a standard curve) or visually (as before, b y use o f solutions o f varying dye concentration to seek the m a x i m u m dye concentration for w h i c h no residual color is left i n the aqueous phase after the extraction). Because the complexation exhibits 1:1 stoichiometry, this m a x i m u m dye


530



concentration s h o u l d b e equal to the p o l y m e r (anionic residue) concentration i n the solution. A g a i n this m e t h o d requires that a strong complex is f o r m e d between the dye a n d the p o l y m e r so that essentially all o f the anionic sites are o c c u p i e d b y dye molecules at t h e equivalence point. A n additional advantage o f the extraction m e t h o d is that it only requires a strong complex f o r m between the dye a n d the polymer, a n d it does not require a spectral shift o r color change. Polyelectrolytes complex w i t h some dyes that exhibit m i n i m a l spectral changes. I f these dyes f o r m strong e n o u g h complexes, they w i l l also be viable candidates for an analytical m e t h o d based o n the extraction process.

Summary and Conclusions Metachromasy, the color-changing ability o f certain ionic dyes i n the pres ence o f oppositely charged polyelectrolytes, has b e e n used to study the s t r u c t u r e - p r o p e r t y relationships o f water-soluble polymers. C o l o r changes i n these dyes, detected b o t h visually a n d spectrophotometrically, have b e e n shown to b e sensitive to their molecular environments a n d states o f aggrega tion as they adhere to p o l y m e r surfaces. T h e s e metachromatic color changes have b e e n correlated to p o l y m e r charge density, degree o f ionization, h y drophobicity, conformation, m o l e c u l a r weight, tacticity, a n d chain flexibihty. T h e effect is fairly nonspecific, however, a n d care must b e taken w h e n the results are interpreted. M e t a c h r o m a s y also c a n b e u s e d as a n analytical tool to measure the concentration o f polyelectrolyte i n aqueous solution. B y quantitatively m o n i toring the spectral changes i n d u c e d i n a metachromatic dye w i t h various concentrations o f polyelectrolyte, a standard curve c a n b e constructed. T h e m e t h o d is fairly sensitive to p H , temperature, i o n i c strength, various i m p u r i ties, a n d interaction time, however, so care must be taken to control all other experimental conditions so that a h i g h degree o f precision c a n b e obtained. T h e color-array a n d the complexation-extraction methods p r o p o s e d h e r e i n are extensions o f the metachromatic methods for quantitative analysis o f polymers. T h e s e methods are based o n the 1:1 stoichiometry o f metachro masy rather than o n the development o f a standard curve a n d so are b e l i e v e d to b e easier to use a n d inherently m o r e accurate than traditional techniques. F u r t h e r m o r e , the color-array m e t h o d is amenable to a visual determination o f p o l y m e r concentration d o w n to 1 p p m , w h i c h obviates the n e e d f o r a spectrophotometer i n field determinations. T o l u i d i n e b l u e Ο showed the most p r o m i s e as a dye f o r use i n t h e color-array m e t h o d . T h e advantage o f the complexation-extraction m e t h o d is that it m a y be a p p l i e d to determine the concentration o f anionic polyelectrolyte u s i n g a nonmetachromatic dye. A s l o n g as a strong neutral complex forms between the cationic dye a n d the poly(acrylate-co-acrylamide) a n d this complex is extractable b y a nonaqueous solvent such as T C T F E , the complexation-extraction m e t h o d m a y b e used


19.

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531

Metachromasy

for the quantitative analysis o f the polymer. T h i s m e t h o d m a y also b e a p p l i e d visually, b u t m o r e accurate results w i l l b e achieved using a spectrophotome ter. T h e d y e Janus green Β showed t h e most promise f o r use i n the complexation-extraction m e t h o d .


Acknowledgments T h e author is grateful to T h o m a s C . M c G o w e n for carrying out m u c h o f the experimental w o r k a n d to N a l c o C h e m i c a l C o m p a n y f o r p r o v i d i n g poly(aerylate-co-aerylamide) samples.

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for review July 15, 1991. ACCEPTED revised manuscript August 10,


Metachromasy: The Interactions between Dyes and Polyelectrolytes in

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