molecules
A icle
How Humic Acids A ec he Rheological and
T anspo P ope ies o Hyd ogels
Ma ina Klucako a * , Ji i Smilek and Pe Sedlacek
Ma e ials Resea ch Cen e, Facul y o Chemis y, B no Uni e si y o Technology, Pu kyno a 118/464,
612 00 B no, Czech Republic; [email p o ec ed].cz (J.S.); [email p o ec ed].cz (P.S.)
*Co espondence: [email p o ec ed].cz; Tel.: +42-054-114-9410
Academic Edi o s: F ancesca D’Anna, Sal a o e Ma ullo and Ca la Rizzo
Recei ed: 5 Ap il 2019; Accep ed: 18 Ap il 2019; Published: 19 Ap il 2019


Abs ac :
Humic acids a e o en ega ded as subs ances wi h a sup amolecula s uc u e which plays
an impo an ole in Na u e. Thei addi ion in o hyd ogels can a ec hei beha io and unc ioning
in di e en applica ions. This wo k is ocused on he p ope ies o widely-used hyd ogel based
on aga ose a e addi ion o humic acids– he p o ona ed H- o m o humic acids and humic acids
wi h me hyla ed ca boxylic g oups. Hyd ogels en iched by humic acids we e s udied in e ms o
hei iscoelas ic and anspo p ope ies. Ro a ional heome y and me hods employing di usion
cells we e used in o de o desc ibe he in luence o humic acids on he p ope ies and beha io o
hyd ogels. F om he poin o iew o heology he addi ion o humic acids mainly a ec ed he loss
modulus co esponding o he elaxa ion o hyd ogel connec ed wi h i s low. In he case o di usion
expe imen s, he anspo o dyes (me hylene blue and hodamine) and me al ions (coppe and nickel)
h ough he hyd ogel was a ec ed by in e ac ions be ween humic acids and he di usion p obes.
The ime lag in he hyd ogel en iched by humic acids was p olonged o coppe , me hylene blue
and hodamine. In con as , he p esence o humic acids in hyd ogel sligh ly inc eased he mobili y
o nickel. The s onges in luence o he me hyla ion o humic acids on di usion was obse ed o
me hylene blue.
Keywo ds: humic acid; hyd ogel; seconda y s uc u e; heology; di usion
1. In oduc ion
Humic acids ep esen na u al biological high-mine alized compounds o igina ed om deg aded
o ganic ma e as he p oduc o animal and plan issue decay [
1
–
3
]. Thei s uc u e is e y complex and
many esea ch eams a e ocused on i s in es iga ion. Ne e heless, he concep o humic acids ha ing a
sup amolecula s uc u e is widely accep ed. Acco ding o he sup amolecula hypo hesis [
1
–
6
], humic
subs ances a e associa ions o small molecules sel -assembled by weak o ces and hyd ogen bonds.
Such associa ions in solu ion a e o med by he sel -o ganiza ion o hyd ophobic and amphiphilic
compounds and a e isola ed p og essi ely om he wa e ne wo k [
6
]. These seconda y s uc u es o
humic subs ances can be dis up ed by he addi ion o o ganic acids [
6
–
9
] o me al ions [
10
]. Fische [
11
]
s a ed ha humic sup amolecula s uc u es ha e pola su aces and unpola co es in soil. This
means ha highly pola subuni s o humic subs ances a e in con ac wi h soil solu ion, ha less
pola subuni s a e in subsequen laye s, and ha unpola subuni s a e inaccessible in he co e o he
humic sup amolecula s uc u e. Nebbioso and Piccolo [
12
] de eloped a “humeomics” app oach
o cha ac e ize he s uc u e o humic subs ances based on sequen ial chemical ac iona ion. O he
au ho s [
9
,
13
–
17
] ha e con i med ha sup amolecula associa ions and mac omolecules can co-exis in
he s uc u e o humic acids.
Al hough, he s uc u e o humic subs ances has been in ensi ely s udied, opinions a e di ided.
On he one hand, i is clea ha he con o ma ional a angemen o humic subs ances can con ol hei
Molecules 2019,24, 1545; doi:10.3390/molecules24081545 www.mdpi.com/jou nal/molecules
Molecules 2019,24, 1545 2 o 13
in e ac ions wi h o he componen s in Na u e [
1
,
10
,
12
,
18
,
19
]; howe e , hese p ocesses a e s ill no
well unde s ood, pa ially as a consequence o he ambiguous complex s uc u e o humic subs ances
hemsel es. The in es iga ion o he seconda y/sup amolecula s uc u e o humic subs ance is
ealized mainly in solu ions. Ou p e ious s udies [
20
–
22
] based on he non adi ional me hods o
high esolu ion ul asound spec ome y, dynamic ligh sca e ing, and mic o- heology showed ha
he molecula o ganiza ion o humic acids in aqueous solu ions could be di ided acco ding o h ee
concen a ion anges. Rea angemen s we e obse ed a concen a ions o a ound 0.02 g
·
dm
−3
and
1 g
·
dm
−3
. Changes in he measu ed alues obse ed a a ound 0.02 g
·
dm
−3
we e less no iceable and
we e ela ed o he o ma ion o pa icles o be ween 100 and 1000 nm in size and changes in he
hyd a ion shells o humic pa icles esul ing in changes in hei elas ici y. The “swi ch-o e poin ”
a a ound 1 g
·
dm
−3
indica ed changes in he seconda y s uc u e o humic acids connec ed wi h an
inc ease in colloidal s abili y, a dec ease in polydispe si y, and minimum alues o iscosi y. The
agg ega ion o humic pa icles and he o ma ion o igid s uc u es in sys ems wi h concen a ions
highe han 1 g
·
dm
−3
we e de ec ed [
20
–
22
]. Many o he au ho s [
5
,
23
–
27
] obse ed a simila b eak in
hei esul s a a ound 1 g
·
dm
−3
and conside ed i as a “swi ch-o e poin ” in he seconda y s uc u e
o humic acids, concluding ha he appa en con o ma ion o humic acids a highe concen a ions
migh di e signi ican ly om he con o ma ion in mo e dilu e sys ems. Ch is l e al. [
25
] s a ed ha
he high-concen a ion egion should co espond o a pseudo-micella o ganiza ion phase. Ou wo ks
also showed ha solu ions p epa ed by dilu ion had di e en p ope ies han solu ions p epa ed
di ec ly a he gi en concen a ion.
Humic subs ances a e ound in di e en en i onmen s (soil, wa e , coal, pea , sedimen s, e c.)
and in a ious, mos ly colloidal, o ms (sol, suspension, gel) [
28
]. Humic sols can be expec ed o
occu p e e ably in wa e sys ems and o ha e all o mos o hei unc ional o eac i e g oups ee
o in e ac . Humic gels migh be ound in wa e sedimen s, swelled pea , soil, o coal. Some o hei
unc ionali ies a e engaged in o ming he gel s uc u e bu he whole sys em, including i s in e io , is
s ill accessible o a ious in e ac ing pa icles, pene able h ough he gel ne wo k. In suspensions,
humic cons i uen s a e igh ly bonded in solid ac ion, and essen ially only he ou e su ace (including
he su ace o po es) is accessible o in e molecula in e ac ions. In iew o he abo e-desc ibed speci ic
complex s uc u e o humic subs ances and hei common occu ence in gel o m, hyd ogel wi h
inco po a ed humic acids was chosen S udies o humic hyd ogels o hyd ogels en iched by humic
subs ances a e ela i ely sca ce. Vashu ina e al. [
29
] added humic acids o s a ch-based hyd ogels
and achie ed a echnically impo an imp o emen in hei heological p ope ies. Zielinska e al. [
30
]
s udied he pa i ioning o humic acids be ween solu ion and hyd ogel. Two ypes o hyd ogels we e
co e ed by humic solu ion, and he pene a ion o humic acids in o hyd ogels was moni o ed. A
nonhomogeneous spa ial dis ibu ion o humic acids in hyd ogels and an accumula ion o humic acids
in a hin ilm on he hyd ogel su ace we e obse ed. Chen e al. [
31
] p epa ed hyd ogel beads om
a s a ch-humic composi e as a biodeg adable adso ben . Thei adso p ion capaci y was highe han
ha o simple s a ch hyd ogel. Kanmaz e al. [
32
] embedded humic acids in o chi osan/poly( inyl
alcohol) hyd ogel in o de o de elop a pH-sensi i e hyd ogel. They concluded ha he swelling o
hyd ogel was con olled by he di usion o sol en in o po es and ha he combina ion o humic
acids wi h hyd ogel had a syne gic e ec . Hyd ogel con aining humic acids exhibi ed smalle -sized
open cells wi h in e connec ed na ow spaces, which made i mo e a ac i e o sol en di usion.
Ma e al. [33]
assumed a double ne wo k in hyd ogels based on polyac ylamide and humic subs ances.
I s adso p ion capaci y and kine ics o di e en me al ions and hei mix u es we e s udied. Se e al
di e en binding si es in he hyd ogel s uc u e we e iden i ied. Hou e al. [
34
] p epa ed an injec able
aga ose hyd ogel inco po a ing sodium huma e and doxo ubicin as an agen o umo managemen
based on he chemo-pho o- he mal he apeu ic e ec .
Ou p imal expe iences [
28
,
35
–
40
] wi h humic hyd ogel a e connec ed wi h he de elopmen o
me hods conce ning he eac i i y and anspo mapping o me al ions in na u al sys ems con aining
humic subs ances. We belie e ha he anspo p ope ies and a es o in e ac ion, ei he co alen o
Molecules 2019,24, 1545 3 o 13
non-co alen , o humic subs ances, a e as impo an as s uc u al issues in e alua ing and unde s anding
he ole o such subs ances in na u al sys ems and human-d i en applica ions. T anspo p ocesses,
including di usion and chemical in e ac ion, a e he e o e impo an o unde s anding he complex
beha io o na u al sys ems and he ole o hei cons i uen s. In ou p e ious wo ks [
28
,
35
–
40
],
hyd ogels we e used as a model o na u al sys ems con aining humic subs ances. The hyd ogels we e
p epa ed by means o he p ecipi a ion o humic acids dissol ed in an alkaline solu ion by s ong acid,
ollowed by washing and cen i uga ion. They we e physically bonded i e e sible hyd ogels wi h
wa e con en s o be ween 80 and 90% w . I was ound ha he anspo o me al ions was s ongly
a ec ed by hei in e ac ions wi h humic acids [
28
,
35
–
40
]. In o de o in es iga e he ole o ca boxylic
g oups in his p ocess, we blocked hem by me hyla ion and in es iga ed he anspo o me al ions in
hyd ogels based on a mix u e o me hyla ed and non-me hyla ed humic acids in di e en a ios [
39
,
40
].
In ou ecen s udies, we ocused on hyd ogels based on aga ose wi h he addi ion o humic acids, he
heological p ope ies o hyd ogels, and he anspo o dyes in hem [
41
–
43
]. Ou p e ious esul s
showed ha a small amoun o humic acids added in o aga ose hyd ogel can signi ican ly a ec he
anspo o dyes in hyd ogels in dependence on he humic con en [
41
,
43
], empe a u e [
41
,
42
], pH,
ionic s eng h [42], and he selec i e blocking o ca boxylic unc ional g oups o humic acids [43].
2. Resul s and Discussion
In his s udy, ou p e ious indings we e summa ized and comple ed by conduc ing new
expe imen s. The anspo o me al ions and o ganic dyes h ough aga ose hyd ogel en iched by
humic acids was s udied alongside he heological cha ac e iza ion o he used hyd ogels. Na i e
isola ed humic acids and hei me hyla ed o m we e s udied in o de o in es iga e he in luence
o humic ca boxylic g oups on he di usi i y o me als and dyes, and he iscoelas ic p ope ies o
hyd ogels. Two me al ions–coppe (II) and nickel(II)—and wo o ganic dyes—me hylene blue and
hodamine—we e chosen as he di usion p obes, because o hei di e en a ini ies o humic acids.
2.1. Di usion Expe imen s
The me hod employing di usion cells was used in ou p e ious s udies [
41
,
43
] and op imized
o di usion h ough aga ose hyd ogel con aining humic acids as he eac i e/immobiliza ion agen .
Concen a ion changes in bo h (dono and accep o ) compa men s we e moni o ed o e ime.
Examples o expe imen al da a ob ained o he di usion o Cu(II) ions h ough he hyd ogel en iched
by MHA a e shown in Figu e 1. In he i s pe iod, he di usion p obe was anspo ed h ough he
hyd ogel and no changes we e obse ed in he accep o compa men . A e ha , he p obe s a ed o
appea in he accep o compa men and he ini ially ze o concen a ion (c
A0
) g adually inc eased. In
con as , he concen a ion dec ease in he dono compa men was slowe due o he ela i ely high
ini ial concen a ion o he di usion p obe (c
D0
). The i s pe iod o he expe imen can be cha ac e ized
by he ime lag (
L
), which is p opo ional o he hyd ogel hickness (L) and he appa en di usion
coe icien (DL) acco ding o Equa ion (1) [41,43,44]:
L=L2/6DL(1)
I is assumed ha he p obes used pene a ed only in o he po es o hyd ogels illed by solu ion
and ha hei di usion in o he (solid) hyd ogel ne wo k was negligible (p ac ically equal o ze o).
The di usion a e is hus dependen mainly on he shape and eal leng h o he di usion pa hway. I
he hyd ogel con ains an ac i e subs ance able o in e ac wi h he p obe, he anspo h ough he
hyd ogel is a ec ed by he in e ac ions and a p opo ion o he p obe pa icles can be immobilized
by he in e ac ion while passing h ough he hyd ogel. The appa en di usion coe icien D
L
hus
cha ac e izes he anspo h ough he hyd ogel wi h an ini ially ze o concen a ion o he p obe. This
means ha he passing p obe on is in con ac wi h he hyd ogel con aining non-occupied binding
Molecules 2019,24, 1545 4 o 13
si es up o he momen when i a i es a he end o he hyd ogel laye and s a s o pe mea e in o he
accep o compa men .
Molecules 2019, 24, x FOR PEER REVIEW 4 o 13
(a)
(b)
Figu e 1. Expe imen al da a ob ained o coppe (II) di using h ough hyd ogel con aining MHA: (a)
Dec ease in he concen a ion o Cu(II) ions in he dono compa men ; (b) Inc ease in he
concen a ion o Cu(II) ions in he accep o compa men wi h ma ked ime lag
L
.
An o e iew o ime lags and co esponding alues o D
L
is p esen ed in Tables 1–4. A
p olonga ion o lag ime o hyd ogels en iched by HA and MHA in compa ison wi h pu e AG
hyd ogel was obse ed o all di usion p obes excep o Ni(II) ions ( hei di usion was sligh ly
as e in en iched hyd ogels). The ime lags we e much longe o o ganic dyes because o hei sizes.
Simila ly, he alues o D
L
de e mined by means o Equa ion (1) dec eased wi h he p olonga ion o
he p obe’s passage h ough hyd ogels (Tables 1–4). The highes alues o D
L
we e ob ained o
me al ions. Only small di e ences we e obse ed in he case o nickel which co esponded wi h i s
e y low a ini y o humic acids [36].
Table 1. Di usion cha ac e is ics ob ained o he anspo o MB h ough hyd ogels.
Hyd ogel Sample
L
(min) D
L
(10
−10
m
2
.s
-1
) D
E
(10
−10
m
2
·s
−1
) D
L
/
D
0
(-) D
E
/
D
0
(-)
AG 177 3.93 1.69 0.47 0.20
AG-HA 464 1.50 0.12 0.18 0.01
AG-MHA 774 0.92 0.17 0.11 0.02
Table 2. Di usion cha ac e is ics ob ained o he anspo o RH h ough hyd ogels.
Hyd ogel Sample
L
(min) D
L
(10
−10
m
2
·s
−1
) D
E
(10
-10
m
2
·s
−1
) D
L
/
D
0
(-) D
E
/
D
0
(-)
AG 242 2.86 2.01 0.32 0.23
AG-HA 436 1.60 0.39 0.18 0.04
AG-MHA 516 1.38 0.28 0.15 0.03
Table 3. Di usion cha ac e is ics ob ained o he anspo o Cu(II) h ough hyd ogels.
Hyd ogel Sample
L
(min) D
L
(10
−10
m
2
·s
−1
) D
E
(10
−10
m
2
·s
−1
) D
L
/
D
0
(-) D
E
/
D
0
(-)
AG 69 10.13 8.63 0.71 0.60
AG-HA 95 7.28 7.87 0.51 0.55
AG-MHA 77 9.03 8.23 0.63 0.58
Table 4. Di usion cha ac e is ics ob ained o he anspo o Ni(II) h ough hyd ogels.
Hyd ogel Sample.
L
(min) D
L
(10
−10
m
2
·s
−1
) D
E
(10
−10
m
2
·s
−1
) D
L
/
D
0
(-) D
E
/
D
0
(-)
AG 70 9.87 2.36 0.75 0.18
AG-HA 57 10.21 2.28 0.77 0.17
AG-MHA 57 10.22 2.29 0.77 0.17
In gene al, when a di usion p obe passes h ough a hyd ogel in o he accep o compa men
and he p obe is hus dis ibu ed h oughou he whole hyd ogel laye , he cha ac e o di usion
Figu e 1.
Expe imen al da a ob ained o coppe (II) di using h ough hyd ogel con aining MHA:
(
a
) Dec ease in he concen a ion o Cu(II) ions in he dono compa men ; (
b
) Inc ease in he
concen a ion o Cu(II) ions in he accep o compa men wi h ma ked ime lag L.
An o e iew o ime lags and co esponding alues o D
L
is p esen ed in Tables 1–4. A p olonga ion
o lag ime o hyd ogels en iched by HA and MHA in compa ison wi h pu e AG hyd ogel was
obse ed o all di usion p obes excep o Ni(II) ions ( hei di usion was sligh ly as e in en iched
hyd ogels). The ime lags we e much longe o o ganic dyes because o hei sizes. Simila ly, he
alues o D
L
de e mined by means o Equa ion (1) dec eased wi h he p olonga ion o he p obe’s
passage h ough hyd ogels (Tables 1–4). The highes alues o D
L
we e ob ained o me al ions. Only
small di e ences we e obse ed in he case o nickel which co esponded wi h i s e y low a ini y o
humic acids [36].
Table 1. Di usion cha ac e is ics ob ained o he anspo o MB h ough hyd ogels.
Hyd ogel Sample L(min) DL(10−10 m2·s−1)DE(10−10 m2·s−1)DL/D0(-) DE/D0(-)
AG 177 3.93 1.69 0.47 0.20
AG-HA 464 1.50 0.12 0.18 0.01
AG-MHA 774 0.92 0.17 0.11 0.02
Table 2. Di usion cha ac e is ics ob ained o he anspo o RH h ough hyd ogels.
Hyd ogel Sample L(min) DL(10−10 m2·s−1)DE(10−10 m2·s−1)DL/D0(-) DE/D0(-)
AG 242 2.86 2.01 0.32 0.23
AG-HA 436 1.60 0.39 0.18 0.04
AG-MHA 516 1.38 0.28 0.15 0.03
Table 3. Di usion cha ac e is ics ob ained o he anspo o Cu(II) h ough hyd ogels.
Hyd ogel Sample L(min) DL(10−10 m2·s−1)DE(10−10 m2·s−1)DL/D0(-) DE/D0(-)
AG 69 10.13 8.63 0.71 0.60
AG-HA 95 7.28 7.87 0.51 0.55
AG-MHA 77 9.03 8.23 0.63 0.58
Table 4. Di usion cha ac e is ics ob ained o he anspo o Ni(II) h ough hyd ogels.
Hyd ogel Sample. L(min) DL(10−10 m2·s−1)DE(10−10 m2·s−1)DL/D0(-) DE/D0(-)
AG 70 9.87 2.36 0.75 0.18
AG-HA 57 10.21 2.28 0.77 0.17
AG-MHA 57 10.22 2.29 0.77 0.17
Molecules 2019,24, 1545 5 o 13
In gene al, when a di usion p obe passes h ough a hyd ogel in o he accep o compa men and
he p obe is hus dis ibu ed h oughou he whole hyd ogel laye , he cha ac e o di usion p ocess
changes. The anspo o he p obe om he dono o accep o compa men s h ough hyd ogel s ill
p oceeds, bu he hyd ogel po es a e illed by he p obe solu ion and an equilib ium be ween eely
mobile p obe ma e ial and immobilized p obe ma e ial can be assumed (i.e., i he hyd ogel con ains
an ac i e subs ance and he p obe can eac wi h i s binding si es). The e ec i e di usion coe icien
DEcha ac e izing his pe iod o di usion can be de e mined as [41,45]:
−βDE =ln [(cD−cA)/(cD0 −cA0)] (2)
whe e βis he so-called cell cons an , i.e., he coe icien cha ac e izing he geome ical pa ame e s o
he expe imen al appa a us. I s alue was de e mined in ou p e ious wo k [
41
] and used in his s udy
o he calcula ion o D
E
. The concen a ions c
D
(dono compa men ) and c
A
(accep o compa men )
belong o a gi en ime ; he ini ial concen a ions a e c
D0
and c
A0
. The alues o D
E
, p esen ed in
Tables 1–4, we e lowe in compa ison wi h he di usion coe icien s D
L
de e mined on he basis o he
ime lag. The di usion coe icien s o dyes (D
E
and D
L
) in AG hyd ogels we e o he same o de o
magni ude as alues published in [46,47]. The e ec i e di usion coe icien s DEdec eased when AG
hyd ogels we e en iched by HA and MHA, bu he di e ences we e again small in he case o nickel.
The di e ences be ween he esul s ob ained o pu e AG hyd ogels and hyd ogels en iched by humic
subs ances can be he esul o wo e ec s. The i s is a possible change in hyd ogel s uc u e. As
desc ibed abo e, he s uc u e o humic acids can be cha ac e ized by a sup amolecula a angemen o
ela i ely small pa icles, e.g., [
6
,
8
,
11
–
13
], o en in co-exis ence wi h bigge mac omolecules [
9
,
13
–
17
].
The s uc u e o humic acids is e y dynamic and sensi i e o ci cums ances (concen a ion, pH, ionic
s eng h) [
21
,
22
]. The e o e, hei inco po a ion in o AG hyd ogel can in luence i s inne s uc u e,
including he dis ibu ion, size and shape o hyd ogel po es. Simila e ec was obse ed in e . [
32
],
whe e chi osan/poly ( inyl alcohol) hyd ogel con aining humic acids exhibi ed smalle -sized open
cells wi h in e connec ed na ow spaces, which made i mo e a ac i e o sol en di usion.
The second e ec is possible in e ac ion be ween he di usion p obes and HA and MHA du ing
he anspo o he p obes h ough he hyd ogel. A he beginning o he di usion expe imen , he
passing p obe on is in con ac wi h hyd ogel con aining non-occupied binding si es and can in e ac
wi h new ee ones. When he on a i es a he end o he hyd ogel laye and s a s o pe mea e
in o he accep o compa men , he eac ion be ween he p obe and ac i e si es s ill p oceeds bu he
in e ac ions a e in equilib ium. This means ha he immobiliza ion o he p obe has he same a e
as i s libe a ion om binding si es. The e o e (in his s age), he p obe is di used h ough hyd ogel
which is sa u a ed and (unde ideal ci cums ances) such anspo can be s a iona y wi h a cons an
di usion a e cha ac e ized by he linea dependence o cAon ime (Figu e 1).
I i is assumed ha di usion p obes do no in e ac wi h pu e AG hyd ogel, he ime lag
de e mined o AG hyd ogel (and he co esponding alue o he appa en di usion coe icien D
L
)
a e gi en only by he combina ion o he po e s uc u e o hyd ogels and he size and shape o he
di usion p obes. A s uc u e pa ame e (
µ
) can be de ined by means o he po osi y (
φ
) and o uosi y
(τ) o he hyd ogel [28,37–43]:
µ=φ/τ. (3)
The alue o
µ
can be de e mined as he a io be ween he di usion coe icien o he p obe in AG
hyd ogel and he di usion coe icien o he p obe in wa e (D
0
). The alues o D
0
o me al ions a e
abula ed [
48
]: 1.43
×
10
−9
m
2·
s
−1
o Cu(II) and 1.32
×
10
−9
m
2·
s
−1
o Ni(II). The alues o D
0
o dyes
we e de e mined in ou p e ious s udy [
42
]. They we e ex apola ed o 25
◦
C and used in his wo k
as: 8.42
×
10
−10
m
2·
s
−1
o MB and 8.93
×
10
−10
m
2·
s
−1
o RH. These esul s a e in ag eemen wi h
alues de e mined using o he me hods [
48
–
50
]. The a ios D
L
/D
0
and D
E
/D
0
a e lis ed in Tables 1–4.
I we ocus on he a ios ob ained o pu e AG hyd ogel, we can s a e ha hei alues a e lowe o
he second s age o di usion, when he p obe is anspo ed h ough he po es illed by i s solu ion.

Molecules 2019,24, 1545 6 o 13
The decele a ion obse ed in he second s age can hus be connec ed wi h spa ial condi ions and he
po en ial concen a ion dependence o he di usion coe icien .
I AG hyd ogels a e en iched by HA and MHA, in e ac ions be ween hei binding si es and
di usion p obes can p oceed and in luence p obe anspo . The di e ence be ween he di usion
coe icien s ob ained o pu e AG hyd ogel and hyd ogels wi h inco po a ed HA and MHA can be
conside ed as he esul o in e ac ions be ween p obes and humic subs ances (and possible changes
in hyd ogel s uc u e). The di e ence be ween HA and MHA can be seen in e ms o he in luence
o ca boxylic g oups and hose hyd ophobized by me hyla ion. Bo h di usion coe icien s (D
E
and
D
L
) o he di usion o p obes in AG-HA and AG-MHA hyd ogels we e de e mined by he s uc u al
cha ac e is ics o hyd ogels, he po en ial concen a ion dependence o di usion coe icien , and he
in luence o chemical eac ions which can di e in i s and second s ages o he expe imen .
As can be seen om Figu e 2, he e ec o humic subs ances inco po a ed in o hyd ogel was
s onge in he case o he di usion o dyes. Su p isingly, he anspo was in luenced mo e in he
second s age o di usion, when he sys em was equilib a ed and he a es o di ec and e e se eac ions
we e he same. One explana ion o his could be he sizes o dye molecules, which a e bigge in
compa ison wi h me al ions. The e o e, a e binding, hey can educe he e ec i e po e diame e s and
supp ess he di usion. Ano he explana ion could be he highe deg ee o immobiliza ion o dyes,
which means ha hey can be bound no only by nega i ely cha ged unc ional g oups bu also by
hyd op obic s uc u es wi hin humic subs ances. In con as , he anspo o Ni(II) ions was p ac ically
una ec ed by he p esence o humic subs ances in he hyd ogel. The di usion o Cu(II) was a ec ed
mainly in he i s s age o he expe imen . This could be connec ed wi h he high a ini y o coppe o
humic subs ances and he s abili y o he o med complexes, which esul ed in low eac ion a es a e
he achie emen o eac ion equilib ium.
Molecules 2019, 24, x FOR PEER REVIEW 6 o 13
I AG hyd ogels a e en iched by HA and MHA, in e ac ions be ween hei binding si es and
di usion p obes can p oceed and in luence p obe anspo . The di e ence be ween he di usion
coe icien s ob ained o pu e AG hyd ogel and hyd ogels wi h inco po a ed HA and MHA can be
conside ed as he esul o in e ac ions be ween p obes and humic subs ances (and possible changes
in hyd ogel s uc u e). The di e ence be ween HA and MHA can be seen in e ms o he in luence o
ca boxylic g oups and hose hyd ophobized by me hyla ion. Bo h di usion coe icien s (D
E
and D
L
)
o he di usion o p obes in AG-HA and AG-MHA hyd ogels we e de e mined by he s uc u al
cha ac e is ics o hyd ogels, he po en ial concen a ion dependence o di usion coe icien , and he
in luence o chemical eac ions which can di e in i s and second s ages o he expe imen .
As can be seen om Figu e 2, he e ec o humic subs ances inco po a ed in o hyd ogel was
s onge in he case o he di usion o dyes. Su p isingly, he anspo was in luenced mo e in he
second s age o di usion, when he sys em was equilib a ed and he a es o di ec and e e se
eac ions we e he same. One explana ion o his could be he sizes o dye molecules, which a e
bigge in compa ison wi h me al ions. The e o e, a e binding, hey can educe he e ec i e po e
diame e s and supp ess he di usion. Ano he explana ion could be he highe deg ee o
immobiliza ion o dyes, which means ha hey can be bound no only by nega i ely cha ged
unc ional g oups bu also by hyd op obic s uc u es wi hin humic subs ances. In con as , he
anspo o Ni(II) ions was p ac ically una ec ed by he p esence o humic subs ances in he
hyd ogel. The di usion o Cu(II) was a ec ed mainly in he i s s age o he expe imen . This could
be connec ed wi h he high a ini y o coppe o humic subs ances and he s abili y o he o med
complexes, which esul ed in low eac ion a es a e he achie emen o eac ion equilib ium.
(a)
(b)
Figu e 2. Ra ios be ween he di usion coe icien s ob ained o hyd ogels en iched by humic
subs ances (D
L
and D
E
) and pu e AG hyd ogels (D
L
and D
E
): (a) Fi s s age o expe imen —Equa ion
(1); (b) Seconds age o expe imen —Equa ion (2).
2.2. Rheological P ope ies o Hyd ogels
I is no easy o dis inguish be ween he e ec o s uc u e and he e ec o he eac i i y o
hyd ogels on he anspo o di usion p obes. In ou p e ious wo ks [41–43], i was assumed ha
po en ial changes in hyd ogel s uc u e caused by he addi ion o humic subs ances can be
neglec ed. This s udy is ocused mo e on he in luence o humic acids on he s uc u e o hyd ogels
and also on hei anspo p ope ies. Rheological measu emen s p o ided in o ma ion abou he
iscoelas ici y o he s udied hyd ogels and changes in hem caused by he addi ion o HA and
MHA.
Figu e 3 shows he dependencies o he s o age and loss moduli on he s ain a cons an
equency. The s o age modulus G’ p opo ional o he elas ic componen o he hyd ogel dec eased
wi h s ain. The s ong dec ease a he gi en s ain means ha he hyd ogel could no esis he
mechanical s ess and ha i s s uc u e was i e e sibly damaged. The b eakdown o he hyd ogel
Figu e 2.
Ra ios be ween he di usion coe icien s ob ained o hyd ogels en iched by humic subs ances
(D
L
and D
E
) and pu e AG hyd ogels (D
L
and D
E
): (
a
) Fi s s age o expe imen —Equa ion (1);
(b) Seconds age o expe imen —Equa ion (2).
2.2. Rheological P ope ies o Hyd ogels
I is no easy o dis inguish be ween he e ec o s uc u e and he e ec o he eac i i y o
hyd ogels on he anspo o di usion p obes. In ou p e ious wo ks [
41
–
43
], i was assumed ha
po en ial changes in hyd ogel s uc u e caused by he addi ion o humic subs ances can be neglec ed.
This s udy is ocused mo e on he in luence o humic acids on he s uc u e o hyd ogels and also on
hei anspo p ope ies. Rheological measu emen s p o ided in o ma ion abou he iscoelas ici y
o he s udied hyd ogels and changes in hem caused by he addi ion o HA and MHA.
Figu e 3shows he dependencies o he s o age and loss moduli on he s ain a cons an equency.
The s o age modulus G’ p opo ional o he elas ic componen o he hyd ogel dec eased wi h s ain.
Molecules 2019,24, 1545 7 o 13
The s ong dec ease a he gi en s ain means ha he hyd ogel could no esis he mechanical
s ess and ha i s s uc u e was i e e sibly damaged. The b eakdown o he hyd ogel ne wo k was
connec ed wi h he co espondence o he maximum o he loss modulus G” wi h he iscous beha io
o he hyd ogel. In gene al, iscoelas ic ma e ials can elax a an applied s ess by lowing. In he
case o he hyd ogels in ou s udy, he low achie ed a maximum a he momen o he collapse o
he hyd ogel ne wo k. The maximum as well as he dec easing pa o he G’cu e was shi ed o he
le o AG-HA and AG-MHA hyd ogels. This means ha he AG hyd ogel was mo e esis an o
applied s ess han hyd ogels en iched wi h humic subs ances and ha he ne wo ks o he AG-HA
and AG-MHA hyd ogels can easily collapse. C oss-o e poin s (G’ =G”) we e de e mined a s ains
o 21%, 8%, and 5% and moduli o 1.8 kPa, 2.5 kPa, and 1.3 kPa o AG hyd ogel, AG-HA hyd ogel,
and AG-MHA hyd ogel, espec i ely. On he basis o hese esul s, a s ain o 0.05% was chosen o
he measu emen o he equency dependencies o moduli. This s ain lay in he egions o linea
iscoelas ici y o all he s udied hyd ogels and was lowe han he s ain causing he collapse o he
hyd ogel ne wo k.
Molecules 2019, 24, x FOR PEER REVIEW 7 o 13
ne wo k was connec ed wi h he co espondence o he maximum o he loss modulus G’’ wi h he
iscous beha io o he hyd ogel. In gene al, iscoelas ic ma e ials can elax a an applied s ess by
lowing. In he case o he hyd ogels in ou s udy, he low achie ed a maximum a he momen o
he collapse o he hyd ogel ne wo k. The maximum as well as he dec easing pa o he G’cu e
was shi ed o he le o AG-HA and AG-MHA hyd ogels. This means ha he AG hyd ogel was
mo e esis an o applied s ess han hyd ogels en iched wi h humic subs ances and ha he
ne wo ks o he AG-HA and AG-MHA hyd ogels can easily collapse. C oss-o e poin s (G’ = G’’)
we e de e mined a s ains o 21%, 8%, and 5% and moduli o 1.8 kPa, 2.5 kPa, and 1.3 kPa o AG
hyd ogel, AG-HA hyd ogel, and AG-MHA hyd ogel, espec i ely. On he basis o hese esul s, a
s ain o 0.05% was chosen o he measu emen o he equency dependencies o moduli. This
s ain lay in he egions o linea iscoelas ici y o all he s udied hyd ogels and was lowe han he
s ain causing he collapse o he hyd ogel ne wo k.
In Figu e 4, he equency dependencies o he s o age and loss moduli a low cons an s ain
a e shown. I was ound ha he ex en o he elas ici y (co esponding wi h he G’modulus)
inc eased wi h inc easing equency. The G’’ modulus had a maximum a 0.03 Hz o AG-HA
hyd ogel and 0.02 Hz o AG-MHA hyd ogel. No maximum was obse ed o pu e AG hyd ogel
(wi hou humic subs ances). The G’’ cu es a highe equencies had a simila cha ac e ; he G’’
modulus dec eased wi h inc easing equency and he lowing abili y o hyd ogels also dec eased. A
egion o cons an G’’ abo e a equency o 1 Hz was obse ed o pu e AG hyd ogel.
(a)
(b)
Figu e 3. Viscoelas ic p ope ies o AG, AG-HA and AG-MHA hyd ogels in dependence on s ain a
a equency o 1 Hz: (a) S o age modulus G’, p opo ional o he ex en o elas ic componen ; (b) Loss
modulus G’’, p opo ional o he ex en o iscous componen .
(a)
(b)
Figu e 3.
Viscoelas ic p ope ies o AG, AG-HA and AG-MHA hyd ogels in dependence on s ain a a
equency o 1 Hz: (
a
) S o age modulus G’, p opo ional o he ex en o elas ic componen ; (
b
) Loss
modulus G”, p opo ional o he ex en o iscous componen .
In Figu e 4, he equency dependencies o he s o age and loss moduli a low cons an s ain a e
shown. I was ound ha he ex en o he elas ici y (co esponding wi h he G’modulus) inc eased
wi h inc easing equency. The G” modulus had a maximum a 0.03 Hz o AG-HA hyd ogel and
0.02 Hz o AG-MHA hyd ogel. No maximum was obse ed o pu e AG hyd ogel (wi hou humic
subs ances). The G” cu es a highe equencies had a simila cha ac e ; he G” modulus dec eased
wi h inc easing equency and he lowing abili y o hyd ogels also dec eased. A egion o cons an G”
abo e a equency o 1 Hz was obse ed o pu e AG hyd ogel.
The heological da a a e summa ized in Figu e 5, whe e he dependence o he a ios be ween he
loss modulus G” and s o age modulus G’ on s ain and equency de e mined o all s udied hyd ogels
a e plo ed. The G”/G’ a io ep esen s a c i e ion o he “liquid beha io ” o he s udied samples. I
he a io is equal o one, he s o age and loss moduli ha e he same alues and he deg ees o liquid
and elas ic ex en s a e p ecisely balanced. Values highe han one deno e ha he sample beha es
as a iscoelas ic liquid. Liquid beha io was obse ed when he hyd ogels we e s ained abo e a
ce ain limi . In Figu e 5a, his limi was a ound a s ain o 16% o AG hyd ogel, 11% o AG-HA
hyd ogel, and 6% o AG-MHA hyd ogel. A e c ossing his limi , he G”/G’ a io inc eased s ongly,
he hyd ogel ne wo k collapsed, and he samples beha ed like liquids. The beginning o he inc ease
in he G”/G’ a io co esponded wi h he maximum o he G” modulus in Figu e 3b. Figu e 5b shows
he maxima o he G”/G’ a io o he AG-HA and AG-MHA hyd ogels. These maxima ep esen he
Molecules 2019,24, 1545 8 o 13
highes ex en o he liquid beha io o he hyd ogels, bu he alues a e much lowe han one and he
ex en o he elas ic componen p edomina ed a all equencies. The esul s show ha he heological
p ope ies o he hyd ogels we e al e ed by he addi ion o humic subs ances and ha he e ec was
s onge o MHA. The beha io o hyd ogels en iched wi h humic subs ances shi ed owa ds ha o
iscoelas ic liquids. This means ha hyd ogels con aining humic subs ances had a lowe abili y o
esis mechanical s esses, which can be connec ed wi h hei highe pe meabili y.
Molecules 2019, 24, x FOR PEER REVIEW 7 o 13
ne wo k was connec ed wi h he co espondence o he maximum o he loss modulus G’’ wi h he
iscous beha io o he hyd ogel. In gene al, iscoelas ic ma e ials can elax a an applied s ess by
lowing. In he case o he hyd ogels in ou s udy, he low achie ed a maximum a he momen o
he collapse o he hyd ogel ne wo k. The maximum as well as he dec easing pa o he G’cu e
was shi ed o he le o AG-HA and AG-MHA hyd ogels. This means ha he AG hyd ogel was
mo e esis an o applied s ess han hyd ogels en iched wi h humic subs ances and ha he
ne wo ks o he AG-HA and AG-MHA hyd ogels can easily collapse. C oss-o e poin s (G’ = G’’)
we e de e mined a s ains o 21%, 8%, and 5% and moduli o 1.8 kPa, 2.5 kPa, and 1.3 kPa o AG
hyd ogel, AG-HA hyd ogel, and AG-MHA hyd ogel, espec i ely. On he basis o hese esul s, a
s ain o 0.05% was chosen o he measu emen o he equency dependencies o moduli. This
s ain lay in he egions o linea iscoelas ici y o all he s udied hyd ogels and was lowe han he
s ain causing he collapse o he hyd ogel ne wo k.
In Figu e 4, he equency dependencies o he s o age and loss moduli a low cons an s ain
a e shown. I was ound ha he ex en o he elas ici y (co esponding wi h he G’modulus)
inc eased wi h inc easing equency. The G’’ modulus had a maximum a 0.03 Hz o AG-HA
hyd ogel and 0.02 Hz o AG-MHA hyd ogel. No maximum was obse ed o pu e AG hyd ogel
(wi hou humic subs ances). The G’’ cu es a highe equencies had a simila cha ac e ; he G’’
modulus dec eased wi h inc easing equency and he lowing abili y o hyd ogels also dec eased. A
egion o cons an G’’ abo e a equency o 1 Hz was obse ed o pu e AG hyd ogel.
(a)
(b)
Figu e 3. Viscoelas ic p ope ies o AG, AG-HA and AG-MHA hyd ogels in dependence on s ain a
a equency o 1 Hz: (a) S o age modulus G’, p opo ional o he ex en o elas ic componen ; (b) Loss
modulus G’’, p opo ional o he ex en o iscous componen .
(a)
(b)
Figu e 4.
Viscoelas ic p ope ies o AG, AG-HA and AG-MHA hyd ogels in dependence on equency
a a s ain o 0.05%: (
a
) S o age modulus G’, p opo ional o he ex en o elas ic componen ; (
b
) Loss
modulus G”, p opo ional o he ex en o iscous componen .
Molecules 2019, 24, x FOR PEER REVIEW 8 o 13
Figu e 4. Viscoelas ic p ope ies o AG, AG-HA and AG-MHA hyd ogels in dependence on
equency a a s ain o 0.05%: (a) S o age modulus G’, p opo ional o he ex en o elas ic
componen ; (b) Loss modulus G’’, p opo ional o he ex en o iscous componen .
The heological da a a e summa ized in Figu e 5, whe e he dependence o he a ios be ween
he loss modulus G’’ and s o age modulus G’ on s ain and equency de e mined o all s udied
hyd ogels a e plo ed. The G’’/G’ a io ep esen s a c i e ion o he “liquid beha io ” o he s udied
samples. I he a io is equal o one, he s o age and loss moduli ha e he same alues and he
deg ees o liquid and elas ic ex en s a e p ecisely balanced. Values highe han one deno e ha he
sample beha es as a iscoelas ic liquid. Liquid beha io was obse ed when he hyd ogels we e
s ained abo e a ce ain limi . In Figu e 5a, his limi was a ound a s ain o 16% o AG hyd ogel,
11% o AG-HA hyd ogel, and 6% o AG-MHA hyd ogel. A e c ossing his limi , he G’’/G’ a io
inc eased s ongly, he hyd ogel ne wo k collapsed, and he samples beha ed like liquids. The
beginning o he inc ease in he G’’/G’ a io co esponded wi h he maximum o he G’’ modulus in
Figu e 3b. Figu e 5b shows he maxima o he G’’/G’ a io o he AG-HA and AG-MHA hyd ogels.
These maxima ep esen he highes ex en o he liquid beha io o he hyd ogels, bu he alues a e
much lowe han one and he ex en o he elas ic componen p edomina ed a all equencies. The
esul s show ha he heological p ope ies o he hyd ogels we e al e ed by he addi ion o humic
subs ances and ha he e ec was s onge o MHA. The beha io o hyd ogels en iched wi h
humic subs ances shi ed owa ds ha o iscoelas ic liquids. This means ha hyd ogels con aining
humic subs ances had a lowe abili y o esis mechanical s esses, which can be connec ed wi h hei
highe pe meabili y.
(a)
(b)
Figu e 5. The a ios be ween he loss modulus G’’ and s o age modulus G’ de e mined o AG,
AG-HA and AG-MHA hyd ogels: (a) The dependence on he s ain a a cons an equency o 1 Hz;
(b) The dependence on he equency a a cons an s ain o 0.05%.
2.3. Rela ionships be ween T anspo and Rheological P ope ies o Hyd ogels
As desc ibed abo e, he inal con en s o humic subs ances in he hyd ogels we e e y low
(0.01% w ). Howe e , hei in luence on he anspo p ope ies o hyd ogels as well as on hei
iscoelas ici y was no negligible. The e could be wo explana ions o his esul . The i s is ha he
sup amolecula s uc u e o humic subs ances can s ongly a ec he “ unc ioning” o hyd ogels a
e y low con en s. The inc ease in he pe meabili y o Ni(II) ions can be caused by he change in he
heological p ope ies o en iched hyd ogels. Ni(II) ions a e known as me als wi h e y low a ini y
o humic subs ances and hus hei anspo h ough AG-HA and AG-MHA hyd ogel should be
a ec ed only by changes in hyd ogel s uc u e. The addi ion o humic subs ances esul ed in an
inc ease in di usion a e because o he shi in hyd ogel iscoelas ici y mo e owa ds he beha io
o liquids and because o he lowe esis ance o hyd ogels o mechanical s esses and he anspo
o p obes. This posi i e e ec on hyd ogel pe meabili y was eclipsed by a u he p ope y o humic
Figu e 5.
The a ios be ween he loss modulus G” and s o age modulus G’ de e mined o AG, AG-HA
and AG-MHA hyd ogels: (
a
) The dependence on he s ain a a cons an equency o 1 Hz; (
b
) The
dependence on he equency a a cons an s ain o 0.05%.
2.3. Rela ionships be ween T anspo and Rheological P ope ies o Hyd ogels
As desc ibed abo e, he inal con en s o humic subs ances in he hyd ogels we e e y low
(0.01% w ). Howe e , hei in luence on he anspo p ope ies o hyd ogels as well as on hei
iscoelas ici y was no negligible. The e could be wo explana ions o his esul . The i s is ha
he sup amolecula s uc u e o humic subs ances can s ongly a ec he “ unc ioning” o hyd ogels
a e y low con en s. The inc ease in he pe meabili y o Ni(II) ions can be caused by he change
in he heological p ope ies o en iched hyd ogels. Ni(II) ions a e known as me als wi h e y low
a ini y o humic subs ances and hus hei anspo h ough AG-HA and AG-MHA hyd ogel should
be a ec ed only by changes in hyd ogel s uc u e. The addi ion o humic subs ances esul ed in an
Molecules 2019,24, 1545 9 o 13
inc ease in di usion a e because o he shi in hyd ogel iscoelas ici y mo e owa ds he beha io
o liquids and because o he lowe esis ance o hyd ogels o mechanical s esses and he anspo
o p obes. This posi i e e ec on hyd ogel pe meabili y was eclipsed by a u he p ope y o humic
subs ances, namely hei eac i i y. Cu(II) ions as well as bo h he dyes s udied in his wo k ha e high
a ini y o humic subs ances and hei in e ac ions s ongly a ec ed hei di usion h ough hyd ogels.
Ca boxylic g oups play impo an oles in he in e ac ions o coppe wi h humic subs ances [
35
–
43
].
The selec i e blocking o hese g oups by me hyla ion caused an inc ease in he D
L
alue de e mined
o he i s s age o he expe imen , when he passing on o Cu(II) ions was in con ac wi h he
hyd ogel con aining non-occupied binding si es o humic subs ances. I he ca boxylic g oups we e
blocked by me hyla ion, he in e ac ions we e less in ensi e and he anspo h ough he hyd ogel was
accele a ed. A simila e ec was obse ed in he second s age o he expe imen , bu he in e ac ions
we e in equilib ium and hei in luence was sligh ly supp essed. The si ua ion su ounding he
anspo o dyes was mo e complex. They a e bigge in compa ison wi h me al ions, which esul ed in
a slowe passage h ough he hyd ogel. The “mo e liquid” cha ac e o hyd ogels en iched wi h humic
subs ances had a smalle e ec han he in e ac ions o dyes wi h humic subs ances. In con as o
me al ions, dyes, due o hei cyclic condensed s uc u es, can in e ac s ongly wi h he hyd ophobic
domains o humic subs ances. Simul aneously, hey can eac wi h nega i ely cha ged unc ional
g oups (simila ly o me al ions). The me hyla ion o humic subs ances educed hei nega i e cha ge
and suppo ed hei in e ac ions wi h dyes ia hyd ophobic domains including me hyla ed g oups.
This esul ed in he u he decele a ion o he anspo o dyes h ough AG-MHA hyd ogel. This
e ec was less impo an when he dyes a i ed a he end o he hyd ogel laye and s a ed o pe mea e
in o he accep o compa men . The in e ac ions we e equilib a ed in his s age o he expe imen . The
in luence o he mo e liquid cha ac e o AG-MHA hyd ogel can hus cause a gen le inc ease in he MB
di usion a e (and DE alue) in compa ison wi h MB di usion h ough AG-HA hyd ogel.
3. Ma e ials and Me hods
3.1. Chemicals
Aga ose (AG; ou ine use class), CuCl
2
.2H
2
O (p.a.), NiCl
2
.6H
2
O (p.a.), me hylene blue hyd a e
(MB; CI basic blue 9), and hodamine 6G (RH; CI basic ed 1) we e pu chased om Sigma-Ald ich
(P ague, Czech Republic).
Humic acids (HA; Leona di e s anda d 1S104H) we e pu chased om he In e na ional Humic
Subs ances Socie y (S . Paul, MN, USA). Thei main cha ac e is ics such as elemen al composi ion and
he con en s and p ope ies o acidic unc ional g oups can be ound on he websi e o he In e na ional
Humic Subs ances Socie y (h p://humic-subs ances.o g/). Thei equilib a ed models oge he wi h
co esponding o ce- ield pa ame e s we e p o ided by molecula dynamics simula ions based on
Vienna Soil O ganic-Ma e Modele [51].
Me hyla ed humic acids (MHA) we e p epa ed om HA by means o he ollowing p ocedu e:
1 g o non-modi ied humic acids was mixed wi h 4 mL o CHCl
3
and 2 mL o me hanol. Then, 4 mL
o 2 M solu ion o ime hylsilyldiazome hane in hexane (TMS-DM) we e added. The mix u e was
s i ed con inuously o 2 h on a o ex. Subsequen ly, an addi ional 0.75 mL o TMS-DM was added.
The ob ained MHA we e d ied o 2 h unde a ni ogen a mosphe e and hen o e nigh a 50
◦
C in an
o en [39,40,43]. The composi ion and p ope ies o HA and MHA a e compa ed in [40].
3.2. P epa a ion o Hyd ogels
The p epa a ion o hyd ogels was based on he he mo- e e sible gela ion o AG aqueous solu ion.
AG was dissol ed in deionized wa e o in an aqueous solu ion o HA o MHA, hen hea ed (80
◦
C)
and s i ed o ob ain a anspa en solu ion, and inally sonica ed o emo e gasses. A e wa ds, he
solu ion was pou ed in o a plas ic ing mold ixed be ween wo glass slides. The solu ion solidi ied
g adually o hyd ogel o m by spon aneous cooling ( o labo a o y empe a u e). When he glasses