High-Resolution Ultrasonic Spectroscopy: Looking at the Interpolyelectrolyte Neutralization from a Different Perspective

High-Resolu ion Ul asonic Spec oscopy: Looking a he In e polyelec oly e Neu aliza ion om a Di e en Pe spec i e Tin Klacic,*Adam Jugl, Milosla Peka , and Da o Ko ace ic* Ci e This: Mac omolecules 2023, 56, 1434−1445 Read Online ACCESS Me ics & Mo e A icle Recommenda ions * sı Suppo ing In o ma ion ABSTRACT: In his s udy, he high- esolu ion ul asonic spec oscopy (HR-US) echnique was applied o examine in e polyelec oly e neu aliza ion. The men ioned me hod was es ed on he example o complexa ion be ween poly(allylammonium) ca ions and poly(ac yla e) anions in aqueous solu ions a pH = 7. I was con i med by HR-US ha he ype o i a ion (s epwise o ab up ), he di ec ion o i a ion, and he ype o backg ound sal a ec he ou come o in e polyelec oly e neu aliza ion. The ob ained esul s we e explained on he basis o ul asonic eloci y and a enua ion changes in he con ex o suspension comp essibili y, a pa ame e ha is ex emely sensi i e o molecula o ganiza ion and in e molecula in e ac ions. Mo eo e , he esul s o HR-US measu emen s p o ed o be consis en wi h p e ious esul s ob ained by mo e adi ional me hods such as dynamic ligh sca e ing, mic ocalo ime y, and elec okine ics. This esea ch demons a es ha HR-US is a con enien and eliable me hod ha can be employed o he in es iga ion o in e polyelec oly e neu aliza ion and polyelec oly e- ela ed p ocesses. 1. INTRODUCTION Polyelec oly es a e mac omolecules (e.g., syn he ic polyme s o biopolyme s) wi h ionic o ionizable unc ional g oups ha can ca y posi i e o nega i e cha ges in solu ion. Mixing o opposi ely cha ged polyelec oly es in aqueous solu ion may esul in polyelec oly e complex (PEC) o ma ion. The occu ence o PECs o en goes along wi h phase sepa a ion, esul ing in ei he solid−liquid o liquid−liquid phase sepa a ion. In he o me case, dense polyelec oly e p ecipi a es a e o med, while he la e case esul s in he o ma ion o polyelec oly e complex coace a es wi h liquid- like p ope ies. 1−4 As his sel -assembly p ocesses o many polyelec oly e pai s a e gene ally isoen halpic, i is belie ed ha PECs a e o med due o an inc ease in en opy caused by he elease o s uc u ed wa e molecules and coun e ions associa ed wi h polyme chains. 5−12 The mo phology, s uc u e, composi ion, and physicochemical p ope ies o o med PECs will depend on nume ous assembly pa ame e s such as concen a ion and cha ge densi y o polyelec oly es, 13 empe a u e, pH, and ionic s eng h o he medium. 5,12,14−16 One in e es ing ac o ha also a ec s he s uc u e and p ope ies o PECs is he ype o used backg ound sal . As has been ecen ly shown by ou and Schleno g oup, 7−10 he in luence o suppo ing anions on PEC composi ion and ene ge ics o in e polyelec oly e neu aliza ion can be easonably well co ela ed wi h he posi ion o he coun e - anion in he Ho meis e se ies. The Ho meis e se ies was disco e ed in p o ein p ecipi a ion expe imen s, 17,18 and his se ies classi ies anions and ca ions acco ding o hei inc easing p ecipi a ion powe as ollows: < < < < < < <SCN ClO I NO B Cl F H PO 4 3 2 4 < < < < < < + + + + + + + Li Na K Rb Cs NH (CH ) N 4 3 4 This lis o ions ank-o de ed in e ms o how s ongly hey modula e p o ein solubili y p o ides a pa h o he modi ica ion o PEC p ope ies simply by changing he ype o sal du ing hei p epa a ion. Clea ly, PECs a e e sa ile ma e ials ha could possibly be used in a wide ange o applica ions, such as ma ices o enzyme immobiliza ion, 19 biocompa ible coa ings o d ug deli e y, 20 and ec o s o gene he apy, 21,22 o name a ew. The p ocess o in e polyelec oly e neu aliza ion is commonly s udied by ligh sca e ing me hods, u bidime y, conduc ome y, po en iome y, spec opho ome y, and calo- ime y. 5−13,23 Each o hese expe imen al echniques gi es aluable in o ma ion abou he examined sys em. Po en iom- e y, o example, p o ides in o ma ion on he p opo ion o pai ed monome s by moni o ing coun e ion ac i i y in solu ion. Howe e , i is limi ed o complexa ion eac ions o “pu e” polyelec oly es (wi hou excess coun e ions). Calo im- e y p o ides aluable in o ma ion on he ene ge ics o in e polyelec oly e neu aliza ion. Howe e , as men ioned ea lie , in e polyelec oly e neu aliza ion p ocesses a e mos ly isoen halpic, 5−12 which limi s he applica ion o calo ime y in esea ch. In addi ion o he abo e, measu able changes in Recei ed: No embe 17, 2022 Re ised: Janua y 31, 2023 Published: Feb ua y 14, 2023 A icle pubs.acs.o g/Mac omolecules © 2023 Ame ican Chemical Socie y 1434 h ps://doi.o g/10.1021/acs.mac omol.2c02349 Mac omolecules 2023, 56, 1434−1445 Downloaded ia BRNO UNIV OF TECHNOLOGY on Ma ch 27, 2023 a 12:26:25 (UTC). See h ps://pubs.acs.o g/sha ingguidelines o op ions on how o legi ima ely sha e published a icles. en halpy always indica e ha a chemical p ocess is aking place. The only p oblem is o de e mine which p ocess i is. Me hods ha moni o he sca e ing o ligh and elec opho e ic mobili y o pa icles p o ide aluable in o ma ion on he size and s uc u e o PECs. Howe e , hey a e limi ed o he mola a ios o i an and i and be o e he onse o loccula ion. UV/ is spec opho ome y is sui able o de e mining he p opo ion o opposi ely cha ged monome s in he eac ion p oduc s. Spec opho ome y can also be used o in es iga e he con e sion o me as able p oduc s o equilib ia. 7 Howe e , o pe o m a spec opho ome ic expe imen , i is necessa y o use spec opho ome ically ac i e polyelec oly es such as poly(sodium 4-s y enesul ona e) (PSS) o a leas label he polyelec oly es wi h some kind o a ma ke . F om all o he men ioned, i can be concluded ha he p ocesses o in e polyelec oly e neu aliza ion a e bes s udied by a combina ion o complemen a y echniques, bu e en hen, as shown by Philipp e al., 13 di e en me hods o en indica e di e en “ i a ion endpoin s” o he same pai o polyme s. High- esolu ion ul asonic spec oscopy (HR-US) is a ela i ely no el spec oscopic echnique o ma e ial analysis. A de ailed desc ip ion o his me hod can be ound in ecen ly published e iew pape s. 24,25 B ie ly, his echnique is based on he measu emen s o eloci y and a enua ion (ene gy losses ha include abso p ion and sca e ing con ibu ions) o ul asonic comp ession wa es p opaga ing h ough he analyzed sample. These ul asonic wa es p obe he elas ic p ope ies o ma e ials de e mined by in e molecula in e - ac ions and molecula o ganiza ion. Thus, HR-US o e s a numbe o ad an ages o e adi ional me hods such as UV/ is spec opho ome y. Fo example, i is a nondes uc i e echnique ha does no equi e op ical ma ke s and can be used on concen a ed and opaque samples. Ano he ad an age is ha i is ela i ely easy o change he wa eleng h o he ul asonic wa e. Unlike spec opho ome y whe e he elec o- magne ic wa e o igina es in a ligh sou ce and he e o e needs special e o o ob ain he equi ed spec al pu i y, ul asonic wa es a e gene a ed elec onically. The e o e, a ypical ul asonic spec ome e can co e a b oad ange o equencies ( om 1 o 20 MHz). In addi ion, HR-US measu emen s can be pe o med wi h esolu ion down o 10−5% o ul asonic eloci y in small sample olumes ( ypically 1 mL and down o 0.03 mL) a ambien o ele a ed p essu es o e a b oad empe a u e ange (−40 o 130 °C) and in a ious measu ing egimes such as au oma ic i a ions and measu emen s in low. Also, HR-US measu emen s can be pe o med in di e en media anging om dilu e solu ions o semi-solid ma e ials such as waxes and gels. The e o e, HR-US is a e y e sa ile me hod ha can be used o analyze a b oad ange o molecula p ocesses. Cu en applica ions o his echnique include analysis o agg ega ion, micelliza ion, and gela ion phenom- ena, 26−30 phase diag ams o mic oemulsions, 31 con o ma ional ansi ions in polyme s and biopolyme s, 32 in e ac ions be ween polyelec oly es and su ac an s o amino acids, 33−35 s abili y o emulsions and suspensions, 36−38 polyme adso p- ion on o su aces, 39 enzyma ic hyd olysis o p o eins and ca bohyd a es, 25,40−43 and many o he s. Despi e being employed o he cha ac e iza ion o nume ous chemical and physical p ocesses, he e is a lack in he li e a u e ega ding he po en ial use o HR-US o s udying he o ma ion o polyelec oly e complexes. The aim o his wo k is o use, o he i s ime, high- esolu ion ul asound spec ome y in a i a ion egime o gain a mo e de ailed insigh in o in e polyelec oly e neu aliza ion o poly(allylamine hyd ochlo ide) (PAH) and poly(ac ylic acid) (PAA) in di e en sal media, namely, NaF, NaCl, NaClO4, LiCl, and (CH3)4NCl. To he bes o ou knowledge, he e is no epo published ye on using his me hod o in es iga e he o ma ion o polyelec oly e complexes. In ou p e ious s udy, 9 complexa ion be ween he same pai o mac oions was examined by means o elec okine ics, mic o- calo ime y, and dynamic ligh sca e ing (DLS). I was ound ha he p esence o di e en elec oly es a ec ed he PAH/ PAA in e polyelec oly e neu aliza ion conside ably, leading o ion-speci ic agg ega ion o polyelec oly e pa icles. He e, we expand he esea ch o HR-US measu emen s, which a e discussed also wi h espec o he ea lie s udy. 9 2. EXPERIMENTAL SECTION 2.1. Ma e ials. Poly(allylamine hyd ochlo ide) (Mw≈17.5 kDa) and poly(ac ylic acid) (Mw≈1000 kDa) we e pu chased om Sigma- Ald ich. The monome unc ionaliza ion deg ees ( ), also known as deg ees o subs i u ion, 44 we e de e mined by po en iome ic i a ions wi h s anda dized NaOH (Me ck) and AgNO3(Kemika) solu ions. The alues ob ained we e 0.88 ±0.01 o PAH and 0.97 ± 0.02 o PAA ( o mo e de ails abou he p ocedu e o de e mining , see he Suppo ing In o ma ion). All polyelec oly e solu ion concen a ions we e co ec ed acco dingly o - alues and a e exp essed wi h espec o he monome epea ing uni (deno ed cm in he ex ). The polyelec oly e solu ions used o HR-US and densi y in es iga ions we e p epa ed by dissol ing a ce ain amoun o he polyelec oly e, 3-(N-mo pholino)p opanesul onic acid (MOPS) bu e (Sigma-Ald ich), and a backg ound sal in deionized wa e . Be o e dissol ing hem, PAH and PAA we e d ied a 60 °C, and sal s we e d ied a 110 °C o abou 200 min. To ensu e he maximum cha ge densi y o bo h mac oions, 45 he pH o polyelec oly e solu ions was adjus ed o 7.0 ±0.1 wi h a 1.0 M NaOH solu ion (Pen a). Fo ha pu pose, a pH me e (888 Ti ando, Me ohm) equipped wi h a combined glass mic oelec ode (6.0262.100, Me ohm), p ecalib a ed wi h s anda d bu e s (Hamil on) wi h pH alues o 4.01, 7.00, and 9.21, was used. NaCl, NaClO4, NaF, (CH3)4NCl, and LiCl sal s o analy ical pu i y we e pu chased om Sigma-Ald ich and we e used wi hou u he pu i ica ion excep o (CH3)4NCl. Te ame hylammonium chlo ide (he ea e e e ed o as Me4NCl), a sal o a e ame hylammonium ca ion Me4N+and a chlo ide anion, was pu i ied by ec ys alliza ion om a mix u e o me hanol (Honeywell Riedel-de-Haen) and ace one (Honeywell Riedel-de-Haen) as desc ibed in he li e a u e. 46 The e hanol used o cleaning he ube o he densi ome e was pu chased om Pen a. The deionized wa e used in all expe imen s was p epa ed in a PURELAB Classic pu i ica ion sys em (ELGA) and had an ini ial conduc i i y lowe han 0.056 μS/cm. 2.2. High-Resolu ion Ul asonic Spec oscopy Measu e- men s. High- esolu ion ul asonic spec oscopy measu emen s we e pe o med in a i a ion egime on an HR-US spec ome e (model 102T) equipped wi h a i a ion accesso y p oduced by Ul asonic Scien i ic (I eland). All expe imen s we e ca ied ou a six selec ed equencies (2.8, 5.3, 8.0, 11.7, 12.3, and 14.9 MHz) and 25.00 °C. Tempe a u e con ol was achie ed by a Haake PC300/A28 wa e ba h (The mo Scien i ic), which p o ided a empe a u e s abili y o 0.01 °C. Be o e he measu emen s, each solu ion was degassed by a 5452 miniSpin cen i uge (Eppendo ) o 5 min a 3500 pm. A e ha , he e e ence cell was illed wi h 1 mL o deionized wa e and he sample cell wi h 1 mL o polyca ion o polyanion solu ion. Then, he sample cell was closed wi h a s oppe , which inco po a ed a line o he injec ion o i an solu ion. The i a ion accesso y was equipped wi h a 50 μL Hamil on sy inge and he i an solu ion was injec ed in o he sample cell au oma ically e e y 13 min in s eps con olled by Pump Moni o so wa e (Ul asonic Scien i ic). A e each injec ion, he i and solu ion was igo ously s i ed o 3 min using a double s i ing sys em ha p o ided e ec i e mixing. A e wa d, he Mac omolecules pubs.acs.o g/Mac omolecules A icle h ps://doi.o g/10.1021/acs.mac omol.2c02349 Mac omolecules 2023, 56, 1434−1445 1435 polyelec oly e mix u e was no s i ed un il he nex addi ion o i an . The di e ences in ul asound eloci y and a enua ion be ween he cells we e moni o ed con inuously h oughou he i a ion. Da a a e he e o e epo ed he e in e ms o eloci y di e ence (Δu) and a enua ion di e ence (ΔN) be ween he sample and e e ence cells. =u u u(sample) (wa e ) (1) =N N N(sample) (wa e ) (2) The measu ed da a we e p ocessed using Ti a ion Analysis so wa e ( e sion 4.50.27.35, Ul asonic Scien i ic) in a way ha means alues o ul asonic pa ame e s we e calcula ed based on da a measu ed in he las 100 s be o e he nex i an addi ion. Each measu emen was made a leas in iplica e, and in he ex and ables, a e age alues o measu ed quan i ies wi h hei s anda d e o o he mean a e epo ed. Howe e , o cla i ica ion, only a e age alues o ul asound eloci y and a enua ion a e epo ed in he igu es. The highes s anda d e o s o eloci y and a enua ion we e 0.08 m/s and 0.36 m−1, and he highes ela i e e o s we e 1.1% and 78%, espec i ely ( o examples o i a ion p o iles wi h s anda d e o s, see Figu e S5). The e o e, ep oducibili y was much be e in he case o ul asound eloci y han ha in a enua ion. Howe e , his did no a ec he conclusions o his esea ch because a enua ion was used only as an auxilia y quali a i e pa ame e and only he qui e ep oducible shapes o he measu ed a enua ion cu es we e ele an . The ul asound equency had no e ec on he measu ed da a (Figu e S6); consequen ly, only esul s o 14.9 MHz a e epo ed he e. Also, he e ec o dilu ion o he i and solu ion on measu ed ul asound pa ame e s was negligible (Figu e S7). I should also be men ioned ha isual obse a ion o he i a ed sys ems was also pe o med ou side he HR-US spec ome e in a es ube wi h a bo om s i e , using he same s eps o he i a ion p ocedu e pe o med in he spec ome e o ob ain a be e insigh in o he i a ion p ocess. 2.3. Densi y Measu emen s. The densi y o polyelec oly e solu ions was measu ed a 25.000 °C using a densi ome e DSA 5000 M (An on Paa ). The densi ome e calib a ion was pe o med a 25.000 °C using ai as a s anda d. Be o e he measu emen s, he samples we e degassed as desc ibed ea lie , and hen hey we e ca e ully injec ed in o a U-shaped bo osilica e glass cell so ha no gas bubbles we e p esen . To pe o m he measu emen s, he U-cell was elec onically exci ed o ib a e a i s cha ac e is ic equency. A e he measu emen s, he U-cell was ho oughly cleaned i s wi h 10 mL o a 5.0 M NaClO4solu ion o emo e any esidues o adso bed polyelec oly es and hen wi h a la ge amoun o deionized wa e and e hanol. Densi y measu emen s we e made on h ee samples o he same composi ion in duplica es, and he densi ies o polyelec oly e solu ions p esen ed he e a e a e age alues o six measu emen s ± s anda d e o s o he mean. 3. RESULTS AND DISCUSSION 3.1. Complexa ion o PAH and PAA wi hou he Added Suppo ing Elec oly e: S epwise Ti a ion. Ul asonic eloci y (u) is one o he main ou pu s o HR-US measu emen . Fo liquids, his pa ame e s ongly depends on he densi y (ρ) and adiaba ic comp essibili y (βS) o he sample acco ding o he well-known New on−Laplace equa ion. =u 1 S (3) The comp essibili y is mainly de e mined by he molecula o ganiza ion and in e molecula o ces in he medium. The e o e, ul asonic measu emen s a e usually accompanied by measu emen s o densi y o enable calcula ions o comp essibili y om ul asonic eloci y and densi y o he sample. He e, in Table 1, we p esen he esul s o densi y and eloci y measu emen s ca ied ou wi h solu ions o poly- (allylamine hyd ochlo ide) and poly(ac ylic acid) in he p esence o MOPS bu e . The densi y and ul asonic eloci y o bo h polyelec oly e solu ions a e simila , which means ha he s udied sys ems ha e much he same comp essibili y. These esul s sugges ha he con o ma ion o PAH and PAA polyme chains in solu ion is almos iden ical, as well as he s uc u e o he ionic a mosphe e and he hyd a ion shell a ound cha ged monome uni s. Howe e , i should be poin ed ou ha he bu e concen a ion was 10 imes highe han ha o polyelec oly es o main ain he pH o he solu ions cons an . As a pH = 7.0, app oxima ely hal o MOPS molecules a e p esen in zwi e ionic and ano he hal in he anionic o m, 48 i is o be expec ed ha MOPS molecules a e associa ed wi h bo h PAH and PAA cha ged segmen s. This means ha MOPS molecules bound o polyelec oly e chains can e ec i ely elimina e he elec os a ic epulsions be ween g oups o he same cha ge so ha mac oions adop globula shape con o ma ion. Fu he mo e, he comp essibili y o p epa ed solu ions is p obably mos a ec ed by he s a e o ee bu e molecules because MOPS is in a la ge su plus. As will be e iden om he esul s o HR-US measu emen s made on polyelec oly e solu ions in he p esence o di e en back- g ound sal s, he comp essibili y o he solu ion is o en de e mined by he mos abundan species. To u he expand ou esea ch, he o ma ion o PEC was moni o ed by he HR-US echnique. Fo his pu pose, he in e polyelec oly e neu aliza ion o PAH and PAA in aqueous MOPS bu e solu ions was ca ied ou a pH = 7.0. The ul asonic eloci y and a enua ion o PAH/PAA complexes, p epa ed by s epwise addi ions o PAH solu ion o PAA solu ion and ice e sa, a e shown in Figu e 1. As can be seen in Figu e 1, h ee egions can be iden i iable o he i a ion p o iles measu ed in bo h di ec ions. In he i s egion, eloci y and a enua ion di e ences linea ly inc ease as he i an - o- i and mola a io enla ges. Visually, he mac oion mix u e in his pa changed om clea o milky clouded a e he i s i an addi ion, and hen he u bidi y e ol ed wi h new i an addi ions (Figu e 2). In egion II, which expands e y na owly a ound he equi alence poin , he eloci y and a enua ion di e ences suddenly dec ease wi h he addi ion o i an solu ion. In his pa , he o ma ion o laky whi e p ecipi a es can be obse ed, which sedimen o e ime (Figu e 2). Chollakup and co-wo ke s 49,50 also epo ed ha polyelec oly e complexa ion be ween PAH and PAA in he absence o sal esul s in a p ecipi a e (no he coace a e), independen o he mixing a io, pH (7.0 and 8.6), and he Table 1. Densi y, Ul asonic Veloci y, and Comp essibili y o 0.005 M Polyelec oly e Solu ions a pH = 7.0 (0.05 M MOPS Bu e ) and 25.0 °C a PAH PAA ρ(kg/m3) 1001.07 ±0.02 1001.22 ±0.06 u(m/s) 1502.77 ±0.06 1502.61 ±0.11 βS(10−13 Pa−1) 4423.3 ±0.3 4423.8 ±0.9 a The ul asonic eloci ies we e calcula ed om he measu ed di e ence in eloci y be ween he sample and e e ence cells a 14.9 MHz using he eloci y o pu e wa e a 25.0 °C (u= 1496.687 m/s) om he li e a u e. 47 Mac omolecules pubs.acs.o g/Mac omolecules A icle h ps://doi.o g/10.1021/acs.mac omol.2c02349 Mac omolecules 2023, 56, 1434−1445 1436 molecula weigh o he employed PAA. Finally, he hi d egion o i a ion p o iles is cha ac e ized by a einc ease in ul asound eloci y and a enua ion upon he addi ion o i an solu ion. This pa is somewha simila o he i s egion o he i a ion p o iles, bu he slope o eloci y and a enua ion dependence on he polyme mola a io is sligh ly smalle han ha in he i s egion. Visually, his s age o he i a ion p o ile co esponds o a clea solu ion wi h locs ha ha e been sedimen ed (Figu e 2). Ob ained eloci y i a ion p o ile (Figu e 1a) can be explained easonably well wi hin he amewo k o he New on−Laplace ela ionship (eq 3) by conside ing he sequence o e en s ha occu upon s epwise addi ion o one polyelec oly e in o he solu ion o he o he , as p oposed by Fuoss and Sadek. 51 Fo simplici y, his sequence o e en s is illus a i ely shown o he case o in e polyelec oly e neu aliza ion o PAH wi h PAA in Figu e 2. When he i s d op o PAA is added o PAH solu ion, he polyelec oly es in e ac , o ming a s able colloidal dispe sion. The esul ing polyme agg ega es, deno ed he e as p ima y complexes (o en called nons oichiome ic 52,53 o quasi- soluble 54 complexes), a e compac clus e s c oss-linked by elec os a ic in e ac ions. Since PAH is ini ially in excess, he in e io o hese p ima y complexes is mos ly composed o PAA molecules, while he ex e io con ains posi i ely cha ged PAH chains. O e all, as epo ed ea lie , 9 PAH/PAA p ima y complexes a e posi i ely cha ged, and hei hyd odynamic diame e is app oxima ely cons an o e a wide ange o polyion mola a ios ( < 0.8). The e o e, each subsequen addi ion o i an p ima ily causes he o ma ion o new elec ically cha ged p ima y complexes. Consequen ly, in his pa o he i a ion p o ile ( egion I in Figu e 1a), he ul asonic eloci y, as well as u bidi y (Figu e 2), o he colloid solu ion inc eases. As men ioned be o e, ul asonic eloci y is de e mined by he comp essibili y and he densi y o he medium. Gene ally, he comp essibili y esponse, which is ex emely sensi i e o molecula o ganiza ion and in e mo- lecula in e ac ions, is domina ing. 24 When a polyelec oly e molecule encoun e s a mac oion o opposi e cha ge, he s ong a ac i e ield be ween hem causes elec os a ic binding o hei monome segmen s, and many o he coun e ions a e displaced. A ound displaced ions, new hyd a ion shells composed o less comp essible wa e molecules a e o med, and hus, he comp essibili y o he sys em dec eases. Figu e 1. Veloci y (a) and a enua ion (b) di e ence ob ained by s epwise i a ions o PAH solu ion wi h PAA solu ion (blue squa es) and ice e sa ( ed ci cles) in MOPS bu e (c= 0.05 M) a pH = 7.0, 25.0 °C, and 14.9 MHz equency. The i an - o- i and mola a io o epea ing uni s was calcula ed on he basis o he added olume o i an solu ion (cm= 0.05 M) o i and solu ion (cm= 0.005 M, V0= 1.0 mL). Dashed lines ha e no physical meaning and we e added as guides o he eye. Figu e 2. Illus a ion o he sequence o e en s ha occu upon s epwise addi ion o PAA solu ion in o PAH solu ion accompanied wi h images o he mix u e a di e en PAA- o-PAH mola a ios deno ed . Mac omolecules pubs.acs.o g/Mac omolecules A icle h ps://doi.o g/10.1021/acs.mac omol.2c02349 Mac omolecules 2023, 56, 1434−1445 1437 The e o e, he linea inc ease in ul asonic eloci y in egion I o he i a ion p o ile (Figu e 1a) is an ou come o he o ma ion o compac and s i PAH/PAA p ima y complexes, he elease o wa e and associa ed coun e ions (in ou case MOPS ions) om cha ged polyelec oly e monome s, and he o ma ion o new hyd a ion shells a ound PECs and coun e - ions. As he a io o i an o i and monome s u he inc eases and app oaches equimola alue, he p ima y complex co ona cha ge is compensa ed by i an molecules. Consequen ly, opposi ely cha ged seconda y complexes bea ing he excess o PAA monome s a he su ace a e o med. Thei in e ac ions wi h he p ima y complexes lead o mac oscopic loccula ion and o e a sho pe iod o sedimen a ion o locs (Figu e 2). The loccula ion is accompanied by he educ ion o o al solid su ace a ea and by he expulsion o s i hyd a ion wa e om he su ace o PECs, which inc eases he comp essibili y and dec eases he ul asonic eloci y o he polyelec oly e mix u e. This sha p dec ease in he eloci y is isible in Figu e 1a ( egion II), ega dless o he polyelec oly e addi ion o de . Howe e , i is in e es ing o no e ha he s eep eloci y and a enua ion dec ease in egion II occu s a di e en mola a ios depending on he di ec ion o i a ion. In he case o he PAH o PAA i a ion di ec ion, i appea s a a mola a io o 0.80, and in he case o he opposi e di ec ion, i appea s a a highe mola a io o 0.95. Mo eo e , his s ep dec ease is la ge o he case o PAA addi ion o PAH solu ion han ha o PAH addi ion o PAA solu ion. The obse ed dependency o he loccula ion poin on he di ec ion o i a ion is in good ag eemen wi h he esul s o DLS measu emen s epo ed ea lie , 9 and i was also ound o PECs p epa ed by o he polyelec oly es. 55−57 Mainly, his mixing o de e ec can be explained by misma ches in kine ically con olled monome pai ing. A e all, nons oichiome ic complexes, especially hose ha depend on he o de o mixing, a e a signa u e o kine ic con ol in polyelec oly e associa ion. 8,58,59 O he easons o obse ed i a ion endpoin disc epancy could be associa ed wi h he deg ee o ioniza ion, deg ee o unc ionaliza ion, and molecula weigh o polyelec oly es used in his esea ch (Table 2). Acco ding o he li e a u e, 45 unde condi ions o he pe o med expe imen s (pH = 7.0), PAH has a highe deg ee o ioniza ion han PAA. I means ha ewe polyca ion molecules a e needed o o e cha ge p ima y complexes ha con ain an excess o he polyanion, and hus, phase sepa a ion akes place a lowe mola a ios. I also means ha he mola a io p esen ed in Figu e 1 is no equal o he cha ge a io o polyelec oly es. Howe e , Li e al. 15 ha e ecen ly demons a ed ha polyelec oly e pai ing in PAH/ PAA complexes is no only achie ed by elec os a ic in e ac ions bu also by he hyd ophobic in e ac ions o he alipha ic polyme backbone and he in e polyme hyd ogen bonding o nonionized ac ylic monome uni s. The o me wo in e ac ions a e p onounced especially a acidic pH. The e o e, aking hyd ogen bonding and hyd ophobic in e ac ions in o accoun makes i di icul o ely only on he cha ge a io o exp essing he i a ion endpoin . Fu he mo e, he eal monome a ios can also be di e en om hose gi en in Figu e 1 due o expe imen al e o s in he s anda diza ion o polyelec oly es. On he o he hand, e en i he unc ionaliza ion deg ees o bo h polyme s a e co ec ly de e mined, he un unc ionalized monome s a e andomly dis ibu ed among he PAH and PAA chains. This a ec s he alue o he co ec ed mola a io a which p ecipi a ion occu s, especially in he case o low unc ionaliza ion deg ees. Howe e , he unc ionaliza ion deg ee o he polyca ion and polyanion used in he he ein in es iga ed case is la ge enough (Table 2) ha i s de ia ion om uni y p obably does no a ec he pai ing o cha ged g oups. One should also keep in mind ha he e e sed mixing o de canno be expec ed o yield PECs o he same s uc u e, since PAH used in he ame o ou in es iga ion has a much smalle molecula mass (sho e chains) han PAA, which mus induce somewha di e en oppo uni ies o assembly du ing he PEC o ma ion p ocess. The ecen in es iga ion o PAH/PAA in e polyelec oly e neu aliza ion in solu ion conduc ed by Ga dlund and co- wo ke s 60 suppo s his s a emen . Re u ning o he esul s in Figu e 1a, one can see ha a e he locs a e o med, ul asonic eloci y inc eases upon u he addi ion o i an solu ion ( egion III o i a ion p o ile). Al hough subsequen inco po a ion o i an chains in o he locs may con ibu e o he solu ion comp essibili y and hus o he eloci y, we belie e ha eloci y inc eases wi h new i an addi ions due o he inc easing numbe o ee i an molecules wi h hyd a ion shells composed o less comp essible wa e molecules. In suppo o his conclusion, we s a e he ollowing: Fi s , he slope o he linea dependence o ul asonic eloci y on he mola a io a e he loccula ion poin ( egion III) was no he same as be o e loccula ion ( egion I) when he i an molecules we e inco po a ed in o he polyelec oly e complex. Fu he mo e, in he ea lie s udy, 9 he co esponding eac ion was in es iga ed calo ime ically, and no measu able hea e ec s abo e equi alence we e obse ed. This inding indica es ha i an molecules do no pa icipa e in u he build-up o PECs, as he esul s o calo ime ic measu emen s we e co ec ed o he hea s o i an dilu ion. In he las ew pa ag aphs, i was demons a ed how measu ing ul asonic eloci y du ing he cou se o polyelec- oly e complexa ion by he HR-US spec ome e can p o ide a be e insigh in o his p ocess based on he comp essibili y changes o he sys em. Like ul asonic eloci y, measu ing a enua ion du ing in e polyelec oly e neu aliza ion can be o g ea impo ance o a be e unde s anding o PEC o ma ion. Ul asonic a enua ion ep esen s he ene gy losses in comp essions and decomp essions o he medium in ul asonic wa es, and i is mainly a ec ed by he he e ogenei y o he medium (in his case, his means he o ma ion o colloidal pa icles in a con inuous liquid medium). 25 In a ew sen ences Table 2. Deg ee o Ioniza ion (α), Deg ee o Func ionaliza ion ( ), Weigh -A e age Molecula Weigh (Mw), Deg ee o Polyme iza ion (DP), and Polydispe si y Index (PDI) o Polyelec oly es Used in This Resea ch a PAH PAA α(%) ≈85 ≈65 (%) 88 ±1 97 ±2 Mw(kDa) ≈17.5 ≈1000 DP 187 13 877 PDI 4.3 a Values o αco espond o pH = 7.0 and we e aken om da a published by Choi and Rubne . 45 Deg ees o unc ionaliza ion we e de e mined by po en iome ic i a ions as explained in Sec ion 2. Molecula weigh s o polyme s, deg ees o polyme iza ion, and polydispe si y indexes (excep o PAH) we e p o ided by he supplie . Mac omolecules pubs.acs.o g/Mac omolecules A icle h ps://doi.o g/10.1021/acs.mac omol.2c02349 Mac omolecules 2023, 56, 1434−1445 1438 ha ollow, we will explain he esul s o a enua ion measu emen s ob ained by he HR-US de ice, which a e shown in Figu e 1b. The inc eased a enua ion in egion I o he a enua ion i a ion p o ile e lec s he o ma ion o new he e ogenei ies, i.e., s able nons oichiome ic PAH/PAA nanoassemblies. Then, close o he equi alence poin , a enua ion dec eases s eeply ( egion II). The s ep dec ease co esponds o he coalescence o p ima y and seconda y complexes ha sedimen in he absence o s i ing. As a esul , he mix u e clea s up ( egion II in Figu e 2), and he e a e only se e al unse led pa icles on he way o ul asonic wa e p opaga ion ha accoun o he ene gy losses. This beha io is consis en wi h he esul s o u bidi y measu emen s ca ied ou by Chen e al. 56 They no ed a d op in u bidi y nea he equi alence poin du ing i a ion o poly- (diallyldime hylammonium chlo ide) (PDADMAC) solu ion wi h po assium sal o poly( inylsul onic acid) (PVSK) and explained i by agg ega ion and sedimen a ion o polyme pa icles. A e he sha p dec ease in a enua ion in he icini y o he equi alence poin ( egion II), he a enua ion again mode a ely inc eases wi h new addi ions o he i an . An inc ease in a enua ion in egion III indica es inc easing he e ogenei y o he sys em, as he concen a ion o ee i an molecules ha sca e he ul asonic wa e inc eases. 3.2. Complexa ion o PAH and PAA wi hou he Added Suppo ing Elec oly e: Ab up Ti a ion. A he beginning o he s epwise i a ion, he monome s o i and a e in conside able excess compa ed o he added monome s o i an , i.e., he p ima y complexes con ain excess monome s o i and. As he i a ion p og esses, he ee chains o i and un ou , which leads o he o ma ion o seconda y complexes ha ins an ly loccula e wi h he p ima y ones. I he cha ge in e sion is linked exclusi ely o he excess o one o he polyelec oly es, only seconda y complexes should o m om he p ima y complexes wi hou mac oscopic phase sepa a ion when a la ge excess o i an molecules is suddenly added o he co esponding suspension. To es his hypo hesis by HR- US, posi i e PAH/PAA p ima y complexes we e p epa ed in MOPS bu e (pH = 7.0) by s epwise i an o i and addi ions up o a 0.6 monome mola a io. This p ocedu e was ollowed by he addi ion o he equi alen amoun o i an o achie e a mola a io o 1.6. The esul s o he ab up i an addi ion in excess ollowing he s epwise addi ion p ocedu e a e depic ed in Figu e 3. As can be seen in Figu e 3, he ou come o he ab up ype o PAA o PAH i a ion was di e en om he ou come o he s epwise i a ion expe imen . Al hough he ul asonic eloci y and a enua ion inc eased equally up o a mola a io o 0.6 in bo h ypes o i a ion due o he p ima y complexes o ma ion, no d op in ul asonic pa ame e s nea equi alence was obse ed in he case o ab up i a ion. Ins ead, a e he addi ion o an equi alen amoun o PAA in o a suspension o p ima y PECs a a mola a io o 0.6, he ul asound eloci y and a enua ion inc eased almos linea ly compa ed o he alues be o e his addi ion. Visual inspec ion o PEC suspension a a mola a io o 1.6 e ealed no mac oscopic phase sepa a ion, jus a milky clouded appea ance (inse in Figu e 3a). Judging by he ob ained esul s, i can be s a ed ha he ab up i an addi ion in excess led o he p ima y PEC o e cha ging and hus o he o ma ion o PAH/PAA seconda y complexes. Nega i ely cha ged seconda y complexes did no ans o m in o spa sely soluble p ecipi a es due o elec os a ic s abiliza ion h ough epulsions be ween pa icles. The e o e, a e he ab up i an addi ion, he ul asonic eloci y inc eased due o he coexis ence o igid seconda y complexes and ee PAA molecules in s able colloidal suspension, and he a enua ion inc eases as a consequence o he inc ease in he e ogenei y. In addi ion o he PAH/PAA pai , he o e cha ging o p ima y PECs achie ed by ab up i an addi ion in excess was p e iously obse ed by DLS and elec opho e ic me hods in he case o PDADMAC-PSS and PAH-PSS pai s. 9 I , he e o e, seems ha he phenomenon o cha ge in e sion o p ima y polyelec oly e complexes is uni e sal, jus like he su ace cha ge in e sion ha occu s du ing he deposi ion o each polyelec oly e laye in he p ocess o p epa ing polyelec oly e mul ilaye s (PEMs). 61−64 The desc ibed esul s a e he addi ional s ong e idence o he al eady obse ed 65 co ela ion be ween he p ocess o in e polyelec oly e complexa ion in solu ion and he o - ma ion o PEMs on he su ace. 3.3. Complexa ion o PAH and PAA in Va ious Suppo ing Elec oly es. The complexa ion o PAH and PAA was also s udied by HR-US in NaF, NaCl, NaClO4, LiCl, and Me4NCl aqueous solu ions. The selec ion o hese sal s was based on ema kable anion- and ca ion-speci ic e ec s obse ed ea lie . 9 As in sal - ee expe imen s, i a ions we e Figu e 3. Veloci y (a) and a enua ion (b) di e ence ob ained by ab up i a ion o PAH solu ion (cm= 0.005 M, V0= 1.0 mL) wi h PAA solu ion (cm= 0.05 M) in MOPS bu e (c= 0.05 M) a pH = 7.0, 25.0 °C, and 14.9 MHz equency ( ed ci cles). The esul s o co esponding s epwise i a ion a e gi en o compa ison (blue squa es). The PAA- o-PAH mola a io o epea ing uni s was calcula ed on he basis o added olume o PAA solu ion o PAH solu ion. Dashed lines ha e no physical meaning and we e added as guides o he eye. Image in (a) shows he s able colloidal solu ion o PAH/PAA seconda y complexes ob ained a a mola a io o 1.6. Mac omolecules pubs.acs.o g/Mac omolecules A icle h ps://doi.o g/10.1021/acs.mac omol.2c02349 Mac omolecules 2023, 56, 1434−1445 1439 ca ied ou in MOPS bu e solu ions a pH = 7.0 wi h i and and i an monome concen a ions o 0.005 and 0.05 M, espec i ely. To ensu e comple e subs i u ion o PAH and PAA coun e ions, which coun e balanced he mac oion cha ge in co esponding bu e solu ions wi h he ions o he in oduced suppo ing elec oly e, a conside able excess o sal wi h espec o he bu e was added o polyelec oly e solu ions. Solu ions o bo h polyions in all cases con ained equal concen a ions o he backg ound sal (c= 0.1 M). The sal concen a ion was also kep high, since he in luence o coun e ion ype on he cou se o in e polyelec oly e neu aliza ion can be obse ed solely in concen a ed sal solu ions. 10 Be o e p esen ing he esul s o i a ion expe imen s, he adiaba ic comp essibili y o p epa ed PAH and PAA i and solu ions in di e en suppo ing elec oly es will be conside ed (Figu e 4). As be o e, co esponding comp essibili y alues we e calcula ed om eq 3 based on he measu ed ul asonic eloci y and densi y da a (see Table S1). Se e al impo an conclusions can be d awn om he ob ained esul s. Fi s , he comp essibili y o polyelec oly e solu ions in he p esence o all in es iga ed backg ound sal s is lowe han he comp essibili y o he same solu ions wi hou sal s (Figu e 4). This means ha he p esence o ions in polyelec oly e solu ions causes changes in molecula o gan- iza ion and in e molecula o ces in he solu ion. These s uc u al changes a e p ima ily associa ed wi h he ac ion o ions’ elec ic ield on wa e and changes in hyd ogen bonding among wa e molecules. 66−68 Mo eo e , he obse ed comp essibili y alues a e p ac ically he same o PAH and PAA solu ions p epa ed in he same sal . This ac indica es ha he ions o he suppo ing elec oly e ha e he g ea es in luence on he comp essibili y o solu ions. Tha is no so unexpec ed conside ing ha he e is a 20 imes highe concen a ion o sal in solu ions han he concen a ion o polyme monome uni s. The e o e, he con ibu ion o he dissol ed mac omolecules o he comp essibili y can be neglec ed in u he conside a ion. Also, he ype o back- g ound sal a ec s he comp essibili y o polyelec oly e solu ions. Conside ing he sodium sal s used, he in luence o anions on he comp essibili y o solu ions inc eases in he o de o F−< Cl−< ClO4 −. This ank o de o ions is in acco dance wi h he posi ion o hese ions in he Ho meis e se ies. 17,18 The in luence o ca ions on he comp essibili y o solu ions also ollows he posi ion o he ions in he Ho meis e se ies, so he comp essibili y inc eases in he sequence Me4N+< Na+< Li+. The adiaba ic comp essibili y o he elec oly e solu ion can be use ully in e p e ed in e ms o ions’ con ibu ions o he s uc u e o he wa e ne wo k. In his manne , ion−wa e and wa e −wa e in e ac ions should be dis inguished. 66−68 Cha ged ions in luence adjacen wa e molecule dipoles by causing s ong elec os a ic con ac ion. This phenomenon, usually called elec os ic ion, 69 causes a pa ial loss in he mobili y o wa e molecules loca ed nea ions. As a esul , hyd a ion shells a ound ions a e o med. Gene ally, hyd a ion wa e is less comp essible han bulk wa e . The e o e, solu ions o ions wi h less hyd a ion wa e , i.e., hinne hyd a ion shells, will ha e highe alues o comp essibili y. Howe e , one should no neglec he e ec o hyd ogen bond eo ganiza ion induced by he p esence o ions on he o e all comp essibili y o he solu ion. Acco ding o Ma cus, 70,71 he ex en o hyd ogen bonding in wa e can be sa is ac o ily desc ibed by a nume ical alue o ΔGHB. Tha physical pa ame e ep esen s he a io o s anda d mola Gibbs ee ene gy o ans e o he solu e om ligh (H2O) o hea y (D2O) wa e and he mola di e ence in he hyd ogen bonding ene gies o ligh and hea y wa e . Ions wi h posi i e ΔGHB alues a e known as wa e s uc u e make s, and ions wi h nega i e ΔGHB alues a e known as wa e s uc u e b eake s. In p inciple, solu ions ha con ain la ge ions, which ha e a hin hyd a ion shell and nega i e ΔGHB alues, will be mo e comp essible han solu ions ha con ain small ions, which ha e a hick hyd a ion shell and posi i e ΔGHB alues. In Table 3,ΔGHB alues o ions p esen in PAH and PAA solu ions and he hickness o hei hyd a ion shells a e lis ed. By inspec ion o Table 3, one can see ha he ela i e ela ionship o obse ed anions comp essibili ies (F−< Cl−< ClO4 −) is in acco dance wi h he a ionale explained abo e. Howe e , ha is no he case o ca ions. Solu ions o well- hyd a ed Li+ion ha e he highes comp essibili y alues o all examined sal s (Figu e 4), bu hey should ha e he lowes comp essibili y acco ding o Ma cus o malism. 70−72 One easonable explana ion o his unexpec ed esul (also obse ed by o he s 73,74 ) could be he o ma ion o clus e s in LiCl solu ion. As Thomas and Elcock 75 no iced in molecula dynamics (MD) simula ions, Li+and Cl−ions o m ionic linea clus e s (s ings). The o ma ion o hese clus e s is accompanied by he expulsion o s i hyd a ion wa e molecules o he bulk space. Consequen ly, solu ion’s comp essibili y should inc ease. In e es ingly, he same au ho s ound ha he p esence o bulky Me4N+ca ions in solu ion inc eases he a e age numbe o hyd ogen bonds pe wa e Figu e 4. In luence o di e en backg ound sal s (c= 0.1 M) on adiaba ic comp essibili y o poly(allylamine hyd ochlo ide) and poly(ac ylic acid) solu ions (cm= 0.005 M) a pH = 7.0 (0.05 M MOPS bu e ) and 25.0 °C. Table 3. Thickness o Hyd a ion Shells (Δ ) and ΔGHB Values o Selec ed Ions a ion Δ (pm) ΔGHB F−79 0.08 Cl−43 −0.61 ClO4 −19 −1.01 Li+172 0.28 Na+116 −0.03 Me4N+14 −0.47 a Ion pa ame e s we e aken om da a published by Ma cus. 70,72 Mac omolecules pubs.acs.o g/Mac omolecules A icle h ps://doi.o g/10.1021/acs.mac omol.2c02349 Mac omolecules 2023, 56, 1434−1445 1440 molecule compa ed o he a e age numbe o hyd ogen bonds in pu e wa e . This s uc u e-making beha io o Me4N+ions is no in acco dance wi h he Ma cus classi ica ion o ions 70,71 and could be an explana ion o he obse ed lowe comp essibili ies o Me4NCl solu ions han expec ed. To uly demons a e ha he comp essibili y o LiCl and Me4NCl solu ions is he excep ion a he han he ule, i would be necessa y o de e mine βS alues o a se ies o solu ions con aining di e en ca ions. Un o una ely, his is ou o he scope o his pape because he aim was o explo e how di e en backg ound sal s a ec he o ma ion o PAH/PAA complexes by HR-US. In Figu e 5 one can see eloci y p o iles ob ained by i a ions o PAH solu ion wi h PAA and ice e sa in he p esence o examined sal s. Fo he sake o easy isual compa ison, he cu es ha e been shi ed e ically along he y- axis by adding a cons an so ha all cu es s a a he same poin . As can be seen o all sal s, he ul asonic eloci y changes wi h he mola a io o polyelec oly es in he same ashion as in sal - ee expe imen s. In he beginning, eloci y inc eases linea ly wi h addi ions o i an , hen i suddenly dec eases a ound he equi alence poin , and inally again inc eases. The simila i y be ween i a ion p o iles ob ained in expe imen s pe o med wi h and wi hou suppo ing elec o- ly es indica es ha o med PECs a e mo e likely p esen in he o m o a p ecipi a e han in he o m o a coace a e (p obably due o he low concen a ion o sal in solu ions). This conclusion is well suppo ed by he li e a u e 49,50 and by addi ional op ical mic oscopy in es iga ion. Fo example, in he op ical mic og aphs, we ob ained, o s oichiome ic PECs p epa ed in NaCl solu ion, a dis inguishable o m o p ecipi a e (Figu e S8). Al hough he cu es p esen ed in Figu e 5 look e y simila , he e a e some impo an di e ences be ween hem. Fo example, in some cases, he sha p d op in ul asonic eloci y occu s a lowe mola a ios as compa ed o he “sal - ee” cu e. In he uppe panel o Figu e 5, he anion in luence on he cou se o in e polyelec oly e neu aliza ion is depic ed. In he case o PAA o PAH i a ion, a sudden dec ease in he ul asonic eloci y o posi i ely cha ged PAH/PAA complexes a a mola a io o 0.6 (NaClO4) and a mola a io o 0.9 (NaCl and NaF) can be obse ed. Howe e , his kind o anion- speci ic e ec is no obse ed in he case o i a ion o nega i ely p ima y complexes (PAH o PAA i a ion). The obse ed e ec o anions on he o ma ion o he PECs can be unde s ood as ollows. No mally, a cha ge o polyelec oly e epea ing uni s is balanced ei he by he complemen a y polyelec oly e (in insic cha ge compensa- ion) o by coun e ions (ex insic cha ge compensa ion). 76 The inc ease in he ionic s eng h o polyelec oly e solu ions esul s in a mo e p onounced coun e ion-polyelec oly e ype o binding. 10 The coun e ion-polyelec oly e binding cons an is he one ha exhibi s ion speci ici y, i.e., polyelec oly es and di e en sal ions a e expec ed o in e ac wi h di e en s eng hs. Recen ly, i was ound ha PAH p e e s he binding o weakly hyd a ed oxyanions such as pe chlo a es o e s ongly hyd a ed halide anions (e.g., Cl−and F−). 9 The e o e, i is o be expec ed ha posi i ely cha ged PAH/PAA p ima y complexes p epa ed in he p esence o NaClO4will ha e a highe con en o compensa ed cha ges in he co ona han he same PECs p epa ed in he p esence o NaCl o NaF. This Figu e 5. In luence o di e en backg ound anions (a, b) and ca ions (c, d) o 0.1 M concen a ion on he eloci y di e ence ob ained by s epwise i a ions o PAH solu ion wi h PAA solu ion (a, c) and ice e sa (b, d) in MOPS bu e (c= 0.05 M) a pH = 7.0, 25.0 °C, and 14.9 MHz equency. The i an - o- i and mola a io o epea ing uni s was calcula ed on he basis o added olume o i an solu ion (cm= 0.05 M) o i and solu ion (cm= 0.005 M, V0= 1.0 mL). Dashed lines ha e no physical meaning and we e added as guides o he eye. The esul s o co esponding sal - ee expe imen s a e gi en o compa ison (do ed line). Mac omolecules pubs.acs.o g/Mac omolecules A icle h ps://doi.o g/10.1021/acs.mac omol.2c02349 Mac omolecules 2023, 56, 1434−1445 1441 leads o he seconda y agg ega ion and inally o he loccula ion o posi i e PEC pa icles a lowe mola a ios. On he con a y, when nega i e PEC pa icles a e p epa ed by i a ion o PAA wi h PAH, he e is no such e ec because he su ace cha ge o his ype o complex is no a ec ed by he anions. Ins ead, he excess o PAA monome s a hei su ace is sc eened by Na+ions ha a e common o all in es iga ed solu ions. The he ein-desc ibed HR-US esul s con i m he p e iously epo ed anion-speci ic agg ega ion o PAH/PAA complexes obse ed by DLS measu emen s. 9 In ha s udy, agg ega ion o posi i e complexes was de ec ed by a la ge inc ease in he pa icle size a mola a ios o 0.6 and 0.8 in he p esence o NaClO4and NaCl, espec i ely (Figu e 6a in e 9). Mo eo e , he anion-speci ic agg ega ion o PAH/PAA complexes was u he explo ed by i a ion o posi i e p ima y complexes wi h simple elec oly e solu ions. Compa ed o NaCl, he obse ed alue o c i ical elec oly e concen a ions needed o he onse o posi i e PAH/PAA complex agg ega ion was signi ican ly lowe when he suspension was i a ed wi h NaClO4. The analogous beha io was no iced in he case o posi i e PAH/PSS complexes. 7 Undoub edly, bo h PAH/PAA and PAH/PSS complexes bea an excess o polyca ion monome s a hei su ace, and ClO4 −ions mo e e ec i ely sc een his cha ge han Cl−ions, which esul s in lowe alues o loccula ion endpoin s. Opposi e o he in luence o anions, he e ec o coun e ca ion ype on he PAH/PAA in e polyelec oly e neu aliza ion is no so p onounced, i.e., he obse ed esul s o all in es iga ed suppo ing elec oly es a e ela i ely simila . As can be seen in Figu e 5c,d, he sudden dec ease in ul asonic eloci y o measu emen s pe o med in NaCl, LiCl, and Me4NCl occu s a a simila mola a io as in he case o sal - ee expe imen s, i espec i e o i a ion di ec ion. The obse ed loccula ion a a ela i ely high i an - o- i and a io (≈0.8) indica es ha monome pai ing in hese suppo ing elec oly es p oceeds as e icien ly as in MOPS bu e solu ions. Howe e , by close examina ion o he p esen ed da a, one can see ha he e is a li le shi in he loccula ion onse owa d lowe monome mola a ios in he case when he polyca ion was added o he polyanion solu ion in he p esence o LiCl and Me4NCl. This inding can be in e p e ed as elec oly e- induced nega i e complex agg ega ion. Namely, Li+and Me4N+ions mo e e icien ly sc een he excess o nega i e cha ge a he PEC su ace han Na+ions and hus induce agg ega ion o pa icles. To summa ize, PAH/PAA in e polyelec oly e neu aliza- ion in sal solu ions seems o esul in bo h posi i e and nega i e p ima y complex agg ega ion due o he masking e ec o ions. This e ec leads o asymme ic in e polyelec- oly e neu aliza ion, esul ing in he excess o i and monome s in he ob ained me as able p ecipi a es. PAH seems o bind he examined ions mo e speci ically han PAA. The eason o he obse ed di e ences in polyca ion and polyanion ion-binding abili ies is s ill no ully unde s ood. Howe e , he ac ha anions al e mo e s ongly he o ma ion o bo h PAH/PAA and PAH/PSS complexes han ca ions sugges s ha ion-speci ic e ec s a e mainly ela ed o he ype o ion and no o such an ex en o he ype o polyelec oly e. This s a emen is well suppo ed by a p e ious s udy on PDADMAC/PSS mul ilaye s. 77 While anion e ec s o his sys em become impo an abo e an ionic s eng h o 0.1 M, he ca ion e ec s become impo an a a much highe ionic s eng h o 0.25 M. Ob iously, he g ea e speci ici y o polyca ions owa d anions han ha o polyanions owa d ca ions can he e o e be expec ed i espec i e o he polyion ype. 4. CONCLUSIONS To ob ain a be e insigh in o in e polyelec oly e neu aliza- ion p ocesses be ween poly(allylammonium) ca ions and poly(ac yla e) anions, a ela i ely no el HR-US echnique has been applied. The ob ained esul s we e compa ed wi h he p e ious s udy whe e mo e adi ional me hods (such as DLS and calo ime y) we e used o examine PEC o ma ion. 9 The esul s o HR-US measu emen s ha e con i med ha PAH/ PAA in e polyelec oly e neu aliza ion in he low sal egime unde goes solid−liquid phase sepa a ion and p oceeds as desc ibed in he pionee ing wo k o Fuoss and Sadek. 51 Fi s , posi i ely o nega i ely cha ged p ima y complexes a e o med wi h he sign o su ace cha ge ha depends on he i a ion di ec ion. Then, i he i a ion is pe o med s epwise, opposi ely cha ged seconda y complexes a e gene a ed, and hey eac wi h he p ima y complexes. This leads o mac oscopic loccula ion ha is accompanied by he expulsion o s i hyd a ion wa e om he su ace o PECs. Howe e , i he i an is added o he suspension o p ima y complexes in g ea excess, loccula ion will no occu , bu a he p ima y PECs will o e cha ge. In addi ion, he esul s o HR-US measu emen s made on PAH/PAA polyelec oly e complexes in he di e en sal media ha e emphasized he impo an ole o ions in PEC o ma ion. Coun e ions p esen in he solu ion a e elec os a ically a ac ed by he cha ged polyelec oly e uni s. Consequen ly, his cha ge sc eening causes asymme ic polyme neu aliza ion (one o he polyelec oly es in he o med complexes is p esen in conside able excess) and agg ega ion o p ima y PECs. 7,9,12,16,78 In e es ingly, he polyion mola a io a which loccula ion occu s ma kedly depends on he ype o suppo ing sal , and ha e ec is mo e p onounced in he case o anions. In he end, i can be es ablished ha he esul s ob ained he e and in he p e ious s udy 9 a e in good ag eemen and mos ly in line wi h expec ed alues. Mo eo e , HR-US complemen s he me hods no mally used o s udy PECs wi h addi ional in o ma ion. Fo ins ance, HR-US is no limi ed o he polyelec oly e mola a ios be o e he onse o loccula ion like DLS so we can make u he conclusions abou he p ocess beyond he loccula ion poin . Also, many in e polyelec oly e neu aliza ions a e isoen halpic 5−12 o do no include UV− is ac i e polyme s (like in his esea ch), which limi s he applica ion o calo ime y and spec opho- ome y. In such cases, HR-US can be used as an app op ia e al e na i e. The e o e, i can be concluded ha HR-US is a con enien and eliable new me hod ha can be used o moni o ing in e polyelec oly e neu aliza ion. O e all, he capabili ies o his echnique could make i a aluable ool o u he in es iga ions o p ocesses ela ed o polyelec oly e complexes such as p ecipi a e/coace a e/solu ion phase ansi ions, 1,49,50 in e polyelec oly e exchanges, 79 o glass ansi ions. 80,81 ■ASSOCIATED CONTENT * sı Suppo ing In o ma ion The Suppo ing In o ma ion is a ailable ee o cha ge a h ps://pubs.acs.o g/doi/10.1021/acs.mac omol.2c02349. Mac omolecules pubs.acs.o g/Mac omolecules A icle h ps://doi.o g/10.1021/acs.mac omol.2c02349 Mac omolecules 2023, 56, 1434−1445 1442