Molecules 2022, 27, 1019. h ps://doi.o g/10.3390/molecules27031019 www.mdpi.com/jou nal/molecules
A icle
The E ec o Sup amolecula Humic Acids on he Di usi i y
o Me al Ions in Aga ose Hyd ogel
Ma ina Klučáko á
Facul y o Chemis y, B no Uni e si y o Technology, Pu kyňo a 118/464, 612 00 B no, Czech Republic;
klucako a@ ch. u b .cz; Tel.: +42-054-114-9410
Abs ac : Humic acids a e known as na u al subs ances o a sup amolecula na u e. Thei sel -as-
sembly abili y can a ec he mig a ion o hea y me als and o he pollu an s in na u e. The o ma ion
o me al-humic complexes can dec ease hei mobili y and bioa ailabili y. This s udy ocuses on
me al ions di usion and immobiliza ion in humic hyd ogels. Humic acids we e pu chased om
In e na ional Humic Subs ances Socie y (isola ed om di e en ma ices—pea , soil, leona di e, wa-
e ) and ex ac ed om ligni e mined in Czech Republic. Coppe (II) ions we e chosen as a model
example o eac i e me als o he di usion expe imen s. The model o ins an aneous plana sou ce
was used o expe imen al da a ob ained om moni o ing he ime de elopmen o coppe (II) ions
dis ibu ion in hyd ogel. The e ec i e di usion coe icien s o coppe (II) ions showed he signi ican
dependence on eac ion abili y o humic hyd ogels. Lowe amoun s o he acidic unc ional g oups
caused an inc ease in he e ec i e di usion coe icien . In gene al, di usion expe imen s seem o
ac as a aluable me hod o eac i i y mapping s udies on humic subs ances.
Keywo ds: humic acids; di usion; in e ac ion; sup amolecula ; sel -assembly; coppe
1. In oduc ion
Reac i i y and anspo p ope ies o me al ions a e impo an bo h o e alua ing
and unde s anding he ole o humic acids in na u al sys ems and human-d i en applica-
ions in sol ing hei s uc u al ques ions. Humic acids a e ecognized as a componen o
na u al o ganic ma e ha plays a key ole in he sel -de oxi ica ion o soils and sedi-
men s. Thei sel -assembly and complexa ion abili y can esul in he educ ion o he mo-
bili y o oxic me al ions (and o he pollu an s), biological up ake and bioaccumula ion o
oxic chemicals in plan s as well as he pollu ion o he unde g ound wa e supplies [1–
5].
The s uc u e o humic acids is e y complex. The e a e se e al hypo hesis and mod-
els o p o e his. Nowadays, he concep o humic acids ha ing a sup amolecula s uc-
u e is widely accep ed. This concep p esumes ha humic subs ances a e associa ions o
small molecules sel -assembled by weak o ces and hyd ogen bonds [6–13]. Humic asso-
cia ions a e o med by he sel -o ganiza ion o hyd ophobic and amphiphilic compounds
whe eby hyd ophilic s uc u es a e o med mainly by ca bohyd a e chains and a oma ic
ings, amphiphilic by ionizable unc ional g oups [14,15]. Some esul s [13] showed ha
he associa ions ha e pola su aces and unpola co es, he e o e pola su aces o humic
pa icles (con aining by dissociable unc ional g oups) a e in con ac wi h soil solu ion,
less pola subuni s a e loca ed in he inne laye s, and unpola s uc u es a e accommo-
da ed in he co e o humic associa ion, ou wa d, owa ds he su ounding ee soil en i-
onmen . O he au ho s [11,16–18] s a ed ha sup amolecula associa ions and humic
mac omolecules can co-exis in he s uc u e o humic acids. Many au ho s [1,10,12,18–
22] con i med ha he con o ma ional a angemen o humic acids can con ol hei in e -
ac ions wi h pollu an s in na u e wi hou ega d o hei p e e ed model o humic
Ci a ion: Klučáko á, M. The E ec
o Sup amolecula Humic Acids on
he Di usi i y o Me al Ions in
Aga ose Hyd ogel. Molecules 2022,
27, 1019. h ps://doi.o g/10.3390/
molecules27031019
Academic Edi o s: F ancesca
D’Anna, Sal a o e Ma ullo
and Ca la Rizzo
Recei ed: 30 Decembe 2021
Accep ed: 28 Janua y 2022
Published: 2 Feb ua y 2022
Publishe ’s No e: MDPI s ays neu-
al wi h ega d o ju isdic ional
claims in published maps and ins i u-
ional a ilia ions.
Copy igh : © 2022 by he au ho . Li-
censee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and con-
di ions o he C ea i e Commons A -
ibu ion (CC BY) license (h ps://c e-
a i ecommons.o g/licenses/by/4.0/).
Molecules 2022, 27, 1019 2 o 10
s uc u e. Ou p e ious wo ks [23–26] showed ha he molecula o ganiza ion o humic
acids is s ongly a ec ed by hei concen a ion. Changes in seconda y s uc u e we e ob-
se ed a concen a ions a ound 0.02 g·dm−3 and 1 g·dm−3. Changes obse ed a lowe con-
cen a ion we e a ibu ed o he o ma ion o pa icles o be ween 0.1 and 11 μm in size
and changes in hei hyd a ion shells. Changes in he seconda y s uc u e o humic acids
esul ed in an inc ease in colloidal s abili y, a dec ease in polydispe si y, and he o -
ma ion o humic agg ega es wi h igid s uc u es we e obse ed in sys ems wi h highe
concen a ions (>1 g·dm −3) we e de ec ed. A simila b eak in humic p ope ies a a ce ain
humic con en in he s udied sys em was also obse ed in o he wo ks [8,27–30] and he
high concen a ion egion is o en conside ed o be a pseudo-micella o ganiza ion phase
[28].
The majo i y o published wo k is ocused on humic acids in solu ions. S udies o
humic hyd ogels a e ela i ely sca ce. In ac , mainly hyd ogels based on humic sub-
s ances in combina ion wi h o he ma e ials we e in es iga ed [31–36]. Humic subs ances
we e added in o hyd ogels based on s a ch [29,36], chi osan and poly ( inyl alcohol) [31],
polyac ylamide [32] and aga ose [30,34,35,37,38]. This wo k ocuses on he hyd ogels
based on aga ose en iched by humic acids. The simple me hod, based on he special ac-
cesso y p o iding con olled ine e ical mo emen o he cu e e in he spec opho om-
e e p o iding concen a ion p o iles o di using pa icles in hyd ogel [35,38], was used.
I was ound ha his me hod can be used in se e al expe imen al a angemen s (di usion
couple, di usion om ins an aneous plana , sou ce, di usion om cons an sou ce, e c.).
Simul aneously, he me hod can be used o he eac i i y mapping o humic acids be-
cause he anspo o me al ions (and o he pollu an s) in humic gels is s ongly in lu-
enced by hei eac i i y [2,24,30–40]. Hea y me al ions can be bound o humic acids wi h
di e en bond s eng hs. The esul s can hus p o ide he in o ma ion on hei mobili y
and (bio)a ailabili y. In his wo k, he me hod o he di usion ins an aneous plana
sou ce me hod [39–44] was used. Coppe /II) ion as a adi ional model ion was used be-
cause o i s s ong a ini y o humic acid [1,2,39,40,42].
2. Resul s and Discussion
The basic cha ac e is ics o he humic samples used a e lis ed in Table 1 [45]. I can
be seen ha he highe con en o ca bon has LEHA, and his sample also has he lowes
con en o hyd ogen, oxygen and O/C a io. In con as , i s C/H a io is he highes . A e y
low O/C a io, O con en and high C con en and C/H a io we e also de e mined o
ESHA. The highes H and O con en s we e de e mined o LGHA and SRHA, accompa-
nied by high O/C a ios. In con as , he highes O con en and O/C a io we e de e mined
o NLHA. The con en o acidic unc ional g oups was highes o SRHA, he lowes o
LGHA. Al hough he O/C a io is conside ed as an indica o o he con en o acidic unc-
ional g oups, he o al acidi y does no ully co espond wi h i . The eason is he p esence
o O in o he unc ional g oups (e.g., ca bonyl C=O). The C/H a io is conside ed as an
indica o o a oma ici y. Highe alues show a lowe a oma ici y and highe con en o
single bonds be ween C a oms.
Table 1. Elemen al composi ion and o al acidi y (β) o s udied humic acids (no malized on d y ash-
ee samples) [45].
Sample C (% a .)
H (% a .)
N (% a .)
O (% a .)
C/H O/C β (mmol·g−1)
LGHA 39.07 38.44 1.08 21.41 1.02 0.55 9.00
NLHA 39.81 35.31 0.74 24.14 1.13 0.61 12.19
ESHA 44.34 33.45 2.71 19.51 1.33 0.44 10.15
SRHA 38.64 37.45 0.74 23.17 1.03 0.60 12.85
PPHA 42.36 34.20 2.38 21.06 1.24 0.50 10.92
LEHA 48.18 33.29 0.80 17.73 1.45 0.37 9.77
Molecules 2022, 27, 1019 3 o 10
In his wo k, he e ec o ligni ic humic acids and s anda d humic acids (pu chased
om IHSS) on he di usion o a small amoun o Cu(II) ions in aga ose hyd ogel was
s udied. Coppe (II) is a adi ional model me al ion used o he eac i i y mapping o
humic subs ances [[1,2,39,40,42]. In con as o p e ious wo ks [37,38], he me hod o in-
s an aneous plana sou ce [39–44] was used. In [37], di usion h ough he laye o hyd o-
gel was s udied in he s a iona y s a e. The expe imen al a angemen o dono and ac-
cep o compa men s spaced by he hyd ogel laye was used. Me al ions passed h ough
he laye and he concen a ion o me al ions in bo h compa men was moni o ed. In [38],
hyd ogels we e sa u a ed by me al ions be o e he expe imen . Humic acids we e in
equilib ium wi h coppe (II) and he elease o me al ions om hyd ogels we e s udied.
The di e ence be ween p e ious wo ks [37,38] and he app oach applied in his s udy is
ha only a small, p ede ined amoun o di using pa icles (me al ions) is applied on he
su ace o hyd ogel. The pa icles hen di use in o hyd ogel and hei su ace
concen a ion dec eases (because he sou ce is exhaus ed soon). A equilib ium, he
homogeneous dis ibu ion o me al ions in hyd ogel is achie ed. I means ha he
concen a ion o me al ions is hen cons an and independen o he dis ance om
in e ace. The concen a ion p o ile in equilib ium is hus he line pa allel wi h he x-axis.
In his wo k, a much lowe concen a ion o me al ions we e p esen in he s udied
hyd ogels and he dependence o di usi i y on he con en o di using pa icles in
hyd ogel was explo ed. The ma hema ical model applied on he da a is based on he
p esump ion ha he di usion is no closed o equilib ium, and ha he di usion
ajec o ies a e sho e ha he hyd ogel dimension. Simul aneously, he amoun o me al
ions applied on he in e ace is known and no me al ions a e p esen in hyd ogel a he
onse o he expe imen . The inal solu ion o second Fick’s second law is [43,44]:
= 
e − 
4e 
(1)
and a e loga i hma ion
 =  
e  − 
4e 
(2)
whe e n s ands o he o al mass o di using compound applied in he o m o a na ow
pulse and S is he c oss–sec ion a ea a ailable o he anspo o he compound. The
e ec i e di usion coe icien De can hen be de e mined om he slope o he linea
eg ession o =() [43,44]. The expe imen al da a ob ained o he hyd ogel
en iched by ESHA and he da a linea ized acco ding o Equa ion (2) a e shown in Figu e
1. I can be seen ha he concen a ion o Cu(II) ions a in e ace dec eased and hey
pe mea ed in longe dis ance in o hyd ogel wi h con inuing di usion as expec ed.
Addi ionally, he expe imen al da a dis ibu ion is in a good ag eemen wi h Equa ion (2),
based on he alue o he coe icien s o de e mina ion.
Molecules 2022, 27, 1019 4 o 10
(a) (b)
Figu e 1. The example o expe imen al da a: (a) Concen a ion p o iles in aga ose hyd ogel en iched
by ESHA in di e en imes om he beginning o di usion (6 h—blue, 24 h— ed, 72 h—g een); (b)
The same expe imen al da a linea ized acco ding o Equa ion 2.
The di usion coe icien D o Cu(II) ions in wa e is abula ed [46] and hei alue is
equal o 1.43 × 10−9 m2.s−1. I me al ions di use in hyd ogel, hei mo ion is in luences by i s
po e s uc u e, and he di usion coe icien in hyd ogel Dg is lowe han ha in wa e (D).
I he hyd ogel con ains a eac i e componen (humic acids in ou case), a po ion o he
me al ions can be immobilized and an appa en equilib ium cons an be ween immobi-
lized and ee mo able me al ions (K = cim / c ee) can be included in o he e ec i e di usion
coe icien De .
 = 
(+1) = 
+1=
+1
(3)
whe e he pa ame e

is he a io o he e ec i e di usi e c oss sec ion, which is a ailable
o anspo o Cu(II) ions, o he bulk c oss sec ion. The a ailable c oss sec ion is smalle
han in case o a homogenous ma e ial because he di usion akes place only h ough he
luid- illed po es and oids o humic hyd ogels. Because he po es a e no s aigh , he
di usion akes place mo e e ec i ely o e a longe dis ance han i would in a homoge-
nous ma e ial. The o uosi y τ is a alue cha ac e izing he longe dis ance a e sed in
he po es. The pa ame e μ (=

τ) ep esen s he in luences o he s uc u e o humic hy-
d ogel and i s local geome y in he di usion [37–40]. The s uc u al pa ame e μ hus can
be calcula ed as he a io be ween he di usion coe icien o Cu(II) ions in non eac i e
hyd ogel (aga ose hyd ogel wi hou humic acids) Dg and he di usion coe icien D o
Cu(II) ions in wa e D. The appa en equilib ium cons an K can be exp essed on he basis
o Equa ion (4) as
= 
 −1
(4)
The alue o Dg o pu e aga ose hyd ogel was ound o be 3.71 × 10−10 m2·s−1. The
alues o he e ec i e di usion coe icien De as well as hose o appa en equilib ium
cons an K a e lis ed in Table 2. The con en s o ee and immobilized Cu(II) ions we e
calcula ed on he basis o he alues o appa en equilib ium cons an K, which is he a io
be ween immobilized and ee Cu(II) ions. We know he o al amoun o Cu(II) ions in
Molecules 2022, 27, 1019 5 o 10
hyd ogel (see sec ion Ma e ials and me hods) is equal o he sum o hese wo o ms o
me al ions. Alongside, we know he a io o hese wo o ms and he combina ion o hese
wo ela ionships esul ed in he con en s o ee mo able and immobilized Cu(II) ions in
hyd ogels.
Table 2. E ec i e di usion coe icien s o Cu(II) ions in hyd ogels en iched by di e en humic acids
(De ), appa en equilib ium cons an (K) and con en s o ee mo able and immobilized Cu(II) ions
in hyd ogels.
Sample
10−10 De (m2·s−1)
K (-) F ee Cu(II) (μmol·g−1)
Immobilized Cu(II) (μmol·g−1)
LGHA
2.38 0.56 58.78 32.22
NLHA
1.79 1.08 59.23 63.67
ESHA 2.09 0.78 57.14 44.36
SRHA 0.74 4.02 25.58 102.92
PPHA 2.13 0.66 62.76 46.44
LEHA 3.39 0.10 89.21 8.49
The alues o di usion coe icien s can be compa ed wi h esul s published by o he
au ho s. Scally e al. [47] de e mined Di usion coe icien s o me als and me al complexes
in hyd ogels based on polyac ylamide he commonly used echnique o di usi e g adi-
en s in hin ilms (DGT). Thei alues o Cu(II) ions anged be ween 6.05 and 6.51 × 10−10
m2·s−1 and dec eased s ongly i me al ions we e complexed wi h humic and ul ic acids.
A simila dec ease in di usion a e caused by complexa ion o me al ions wi h humic
subs ances and o he ligands was obse ed in [48]. Wang e al. [49] s udied di usion cha -
ac e is ics o aga ose hyd ogel used in DGT. The di usion coe icien s h ough he aga-
ose gel (6.59 × 10−10 m2·s −1 o Cu) we e sligh ly highe han alues epo ed o he aga-
ose c oss-linked polyac ylamide. Ga mo e al. [50] de e mined he di usion coe icien s
o 55 elemen s in di usi e aga ose polyac ylamide gels o he egula ype used in he
DGT echnique. Thei alues o coppe anged be ween 5.5 and 6.6 × 10−10 m2·s −1. In con-
as , he alues o di usion coe icien s we e lowe in magni ude (e.g., 4.75 × 10−11 m2·s −1
[51] and 2.12–3.12 × 10−11 m2·s −1 [52]). Ou alue o Dg o pu e aga ose hyd ogel is lowe
in compa ison wi h he di usion coe icien s published in [47–50]. I is necessa y o ac-
coun o di e en ypes o hyd ogels and he di e en me hods o he de e mina ion o
he di usion coe icien . Resul s published in [47–50] we e de e mined using he DGT
echnique, which is cha ac e ized by he di usion in hin ilm. The me hod used in his
wo k belongs o he di usion in semi-in ini e medium, whe e he di used pa icles pen-
e a e in o hyd ogel and hei concen a ion on he end o hyd ogel sample is unchanged
du ing he expe imen . The inco po a ion o humic acids in o he hyd ogel esul ed in he
dec ease in di usi i y o a magni ude compa able wi h alues published in [51] and [52].
The abo e men ioned equa ions can be used o da a p ocessing on condi ion ha
he chemical eac ion be ween Cu(II) ions and humic acids is much as e ha he di u-
sion, and he local equilib ium can be assumed o exis be ween he ee and immobilized
me al ions. I means ha he on o di using Cu(II) ions a els h ough he hyd ogel
con aining non-occupied binding si es. Me al ions hus can in e ac immedia ely wi h hu-
mic acids and he quick local equilib ium can be achie ed. On he basis o he knowledge
o o al acidi ies β (see Table 1) and calcula ed alues o appa en equilib ium cons an K,
he con en o ee mo able and immobilized Cu(II) ions in hyd ogels can be de e mined
(Table 2).
As can be seen, he alues o e ec i e di usion coe icien s we e ound o be o he
magni ude ~10−10 m2·s −1 which co esponds wi h alues published elsewhe e [37,38] o
me al ions. In compa ison wi h he esul s ob ained o highe amoun s o di using Cu(II)
ions [38], he e ec i e di usion coe icien s de e mined in his wo k a e lowe , being on
a e age ~30% o he p e ious esul s wi h he excep ion o LGHA (45 %) and SRHA (9 %)
(see Figu e 2). These di e ences be ween he da a epo ed he ein and hose p e iously

Molecules 2022, 27, 1019 6 o 10
ob ained by di e en expe imen al a angemen s and ma hema ical models can be caused
by mul iple ac o s. 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 [12,16,17,53-55] o en in co-
exis ence wi h la ge mac omolecules [11,16–18]. The s uc u e o humic acids is e y dy-
namic and sensi i e o he su ounding en i onmen (concen a ion, pH, ionic s eng h)
[23–25,31,41,53]. The inco po a ion o humic acids in o 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 [37]. Despi e he
ac ha he con en o humic acids is he same o all o he humic samples used and he
same as in p e ious wo ks [34,35,37,38], di e en humic acids ha e di e en s uc u es,
di e en con en o binding si es and di e en molecula a angemen s. The sup amolec-
ula s uc u e o humic acids is e y sensi i e o hei chemical icini y, including he
p esence o eac i e me al ions. Me al ions in luence no only pH and ionic s eng h bu
also can al e he spa ial con igu a ion o humic acids. They ha e mo e possibili ies o
in e ac ing wi h humic acids and coo dina ing mo e binding si es in o one me al-humic
complex. The binding si es can be unc ional g oups belong o one o mo e humic mole-
cules [23,24].
(a) (b)
Figu e 2. Resul s o di usion expe imen s: (a) The compa ison o e ec i e di usion coe icien s de-
e mined in his s udy (blue) and p e ious wo ks (g ey); (b) The con en o ee mobile (g een) and
immobilized ( ed) ac ion o Cu (II) ions in hyd ogels en iched by humic acids.
The mos impo an binding si es in humic molecules a e ca boxylic and phenolic
unc ional g oups [1–6,22,32,39,40]. Thei di e en combina ions can esul in a la ge
numbe o di e en coo dina ion (chela ion) si es ha a e able o bind me al ions by a -
ac i e in e ac ions o di e en s eng hs. The si es can be bi unc ional (e.g., salicylic ype)
o complexes connec ing wo bi unc ional ligands can be o med. A possibili y o o he
ypes (e.g., h ee unc ional g oups) can be also aken in o conside a ion. The spa ial a -
angemen o humic acids and he o ma ion o me al-humic complexes a e hus s ongly
a ec ed by he con en o me al ions in he hyd ogel (o mo e p ecisely by he numbe
a io be ween me al ions and binding si es in humic acids). I esul s in di e en e ec i e
di usion coe icien s and (also) appa en equilib ium cons an s included in he alues o
De (see Equa ion (3)). As men ioned abo e, he alue o K is only appa en . This means
ha he simple equilib ium be ween ee and immobilized ions (Cu (II) ee  Cu (II)im)
does no co espond wi h he eal mechanism o eac ion be ween me al ions and humic
acids in hyd ogel. One o he di e ences can be, e.g., he spli ing o o a hyd ogen ion
om acidic unc ional g oup du ing complexa ion. The e o e, he alue o K canno be
conside ed as he ue equilib ium cons an om a he modynamic s andpoin , bu as he
a io be ween wo o ms o me al ions in hyd ogel ( esembling o a pa i ioning coe i-
cien ). The main di e ences be ween he esul s o his s udy and he alues o De
Molecules 2022, 27, 1019 7 o 10
de e mined by means o o he expe imen al se ups and ma hema ical models a e due o
he dynamic sup amolecula s uc u e o humic acids ha a e able o eac sensi i ely o
changes in hei en i onmen and a po en ial dependence o he di usi i y o me al ions
on hei concen a ion. I is also necessa y o ake in o accoun he in luence o me al ions
on o he condi ions o he su ounding en i onmen , such as pH and ionic s eng h.
By compa ing he pe cen ages o ee mobile and immobilized ac ions o Cu (II)
ions in hyd ogels, i can be s a ed ha only humic acids isola ed om wa e ma ices
(SRHA and NLHA) can immobilize mo e han 50% o me al ions. The immobilized ac-
ion p edomina es only in he case o SRHA and bo h ac ions a e app oxima ely compa-
able o ESHA, NLHA and PPHA. In con as , he ee mobile ac ion p edomina es in
he cases o coal-based humic acids (LGHA and LEHA). Wi h he excep ion o SRHA,
hese indings a e gene ally in ag eemen wi h he esul s epo ed p e iously [38], whe e
he mobile ac ion o Cu (II) ions p e ails in all s udied hyd ogels.
Figu e 3 shows he dependence o he e ec i e di usion coe icien s on he acidi y
o he s udied humic acids. All hyd ogels con aining humic acids had lowe di usi i y in
compa ison o pu e aga ose hyd ogel, as expec ed. The dec ease was caused by he high
a ini y o coppe o humic acids and i s pa ial immobiliza ion in hyd ogel s uc u e. P e-
ious wo ks showed ha he anspo o me al ions in humic gels is s ongly in luenced
by he eac i i y o humic acids; he e o e, changes in he con en o acidic unc ional
g oups esul in changes o di usi i y [39–41]. The in e se p opo ionali y be ween he
e ec i e di usion coe icien s and he con en o acidic unc ional g oups was obse ed.
I indica es ha he inc ease in he con en o acidic unc ional g oups was ela ed o he
dec ease in he mobili y o Cu (II) in hyd ogels.
Figu e 3. The dependence o e ec i e di usion coe icien calcula ed using Equa ion (2) on he con-
en o acidic g oups in humic acids (see β alues in Table 1).
3. Ma e ials and Me hods
Six di e en humic acids we e used in his wo k. One sample o humic acids was
ex ac ed om ligni e mined in he Czech Republic (Mikulčice in Sou h Mo a ia) and he
o he samples we e pu chased om In e na ional Humic Subs ances Socie y (IHSS, S .
Paul, MN, USA) [45].
Ligni ic humic acids (in his wo k designa ed as LGHA) we e isola ed ollowing he
same p ocedu e used in ou p e ious esea ch [2,22–26] (mo e de ails ega ding chemical
s uc u e and he isola ion p ocedu e can be ound elsewhe e [2,34,35,39,49].
The ollowing humic samples we e pu chased om IHSS: No dic Lake Humic Acids
1R105H (NLHA), Ellio Soil Humic Acids 1S102H (ESHA), Suwannee Ri e Humic Acids
Molecules 2022, 27, 1019 8 o 10
2S101H (SRHA), Pahokee Pea Humic Acids 1S103H (PPHA), and Leona di e Humic Ac-
ids1S104H (LEHA).
All hyd ogels u ilized in di usion expe imen s we e p epa ed ia he mo e e sible
gela ion o aqueous solu ion o aga ose desc ibed p e iously [34]. Aga ose hyd ogels
we e gela inized om he solu ion o aga ose in wa e o aqueous solu ions o humic ac-
ids. D y aga ose con en in gel was 1 w .%, d y con en o humic acids was 0.01 w .%. The
mix u e was slowly hea ed unde con inuous s i ing a up o 80 °C and main ained a he
empe a u e un il he occu ence o he anspa en solu ion. The solu ion was degassed
in ul asonic ba h and slowly pou ed in o he PMMA spec opho ome ic cu e e. The
cu e e o i ice was immedia ely co e ed wi h he p e-hea ed pla e o glass o p e en d y-
ing and sh inking o he gel. Gen le cooling o he cu e es a labo a o y empe a u e led
o he g adual gela ion o he mix u e [34,35,37,38].
A squa e slice o il e ing pape (1 × 1 cm) was sunk in o he solu ion o he 1M CuCl2
(1 min) and hen added o one op o he cu e e illed wi h he hyd ogel. The amoun o
Cu (II) ions in he il e ing pape was de e mined as he a e age o measu emen s in
leacha es om en sa u a ed il e ing pape s. The amoun o Cu (II) ions di used in o hy-
d ogel was calcula ed as he di e ence be ween a e age Cu (II) con en in il e ing pape
and he esidue o Cu (II) ions in he il e ing pape a e i s emo ing. The cu e e was
packed wi h pa a ilm and aluminium oil o p e en he hyd ogel d ying. The du a ions
o he di usion expe imen s we e 6, 12, 24, 48 and 72 h. In hese ime in e als, he cu e es
we e aken ou o he solu ion and he UV-VIS spec a we e eco ded conside ing he dis-
ances om he o i ice on Va ian Ca y 50 UV-VIS spec opho ome e (Agilen Technolo-
gies, Palo Al o, CA, USA) equipped wi h he special accesso y p o iding a ine con ol o
he e ical mo emen o he cu e e in he spec opho ome e . The concen a ion o Cu
(II) ions was de e mined a di e en posi ions in he gels by means o a calib a ion line.
UV-VIS spec a we e calib a ed o he hyd ogels wi h he known concen a ion, homog-
enously dis ibu ed in he whole olume o he gel. These hyd ogels samples we e p e-
pa ed using exac ly he same p epa a ion p ocedu e as o he samples o he di usion
expe imen s; only he p ecise amoun o he dye was added o he solu ion be o e gela i-
niza ion. Aga ose (AG; ou ine use class) and CuCl2.2H2O (p.a.) we e pu chased om
Sigma-Ald ich (S . Luis, MO, USA). All expe imen s we e pe o med a labo a o y em-
pe a u e (25 ± 1 °C). Da a a e p esen ed as a e age alues wi h s anda d de ia ion ba s.
4. Conclusions
In his wo k, he in luence o humic acids on he anspo o Cu (II) ions in aga ose
hyd ogels was s udied. I was con i med ha humic acids in luenced he di usi i y o
me al ions in hyd ogels. Di usion coe icien in pu e aga ose hyd ogel (3.71 × 10−10 m2·s−1)
dec eased signi ican ly i he hyd ogel was en iched by humic acids. The lowes alue was
ob ained o he aqua ic SRHA sample, which was also he mos ac i e in coppe -humic
in e ac ions and had he highes con en o acidic unc ional g oups. In con as , a low
con en o unc ional g oups in LEHA sample esul ed in an e ec i e di usion coe icien
compa able wi h he di usi i y o Cu (II) ions in hyd ogel wi hou humic acids. In com-
pa ison wi h some esul s p e iously epo ed, only he small de ined impulse o di us-
ing pa icles pene a ed in o hyd ogel, which led o lowe alues o e ec i e di usion
coe icien s and a highe o compa able immobilized ac ion o cup ic ions (excep ing
LEHA). The di usion was also in luenced by he su ounding en i onmen (pH and ionic
s eng h) as well as he spa ial a angemen o humic acids.
Funding: This esea ch was unded by he Na ional P og amme o Sus ainabili y I (Minis y o
Educa ion, You h and Spo s), g an numbe REG LO1211, Ma e ials Resea ch Cen e a FCH BUT-
Sus ainabili y and De elopmen .
Ins i u ional Re iew Boa d S a emen : No applicable
In o med Consen S a emen : No applicable
Molecules 2022, 27, 1019 9 o 10
Da a A ailabili y S a emen : Expe imen al da a can be p o ided by au ho i equi ed.
Con lic s o In e es : The au ho s decla e no con lic o in e es .
Sample A ailabili y: Samples o he compounds a e a ailable om he au ho s.
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