molecules
A icle
How he Sup amolecula Na u e o Lignohuma e
A ec s I s Di usion in Aga ose Hyd ogel
Ma ina Kluˇcáko á* , Michal Kalina and Voj ˇech Ene
Ma e ials Resea ch Cen e, Facul y o Chemis y, B no Uni e si y o Technology, Pu kyˇno a 118/464,
612 00 B no, Czech Republic; [email p o ec ed].cz (M.K.); [email p o ec ed].cz (V.E.)
*Co espondence: [email p o ec ed].cz; Tel.: +42-054-114-9410
Academic Edi o : Hi oyasu Yamaguchi
Recei ed: 16 Oc obe 2020; Accep ed: 8 Decembe 2020; Published: 10 Decembe 2020


Abs ac :
Lignohuma e, as an indus ially p oduced analog o na u al humic subs ances, is s udied
om he poin o iew o i s di usion p ope ies. This wo k ocuses on i s pe mea ion abili y,
impo an in ag icul u al and ho icul u al applica ions, connec ed wi h i s pene a ion in o plan
o gans as lea es and oo s. The hyd ogel based on aga ose was used as a model ma e ial o he
di usion o lignohuma e. Two ypes o expe imen s we e ealized: he di usion o lignohuma e
in he hyd ogel di usion couple and he di usion o lignohuma e om i s solu ion in o hyd ogel.
The di usion coe icien o lignohuma e in he hyd ogel was de e mined and used o he modelling o
he ime de elopmen o concen a ion p o iles. I was ound ha he model ag ees wi h expe imen al
da a o sho imes bu an accumula ion o lignohuma e in on o he in e ace be ween dono and
accep o hyd ogels was obse ed a e se e al days. The pa icle size dis ibu ion o lignohuma e
and changes in he E
4
/E
6
a io used as an indica o o molecula weigh o humic subs ances
we e de e mined. The esul s showed ha he sup amolecula s uc u e o lignohuma e can eac
sensi i ely o ac ual changes in i s en i ons and hus a ec hei mobili y and pe meabili y in o
di e en ma e ials. A il a ione ec a hein e ace canbe obse edas anaccompanying phenomenon
o he e-a angemen in he lignohuma e seconda y s uc u e.
Keywo ds: lignohuma e; sup amolecula na u e; di usion; sp eading; hyd ogel
1. In oduc ion
Lignohuma e is an indus ially p oduced analog o na u al humic subs ances, p oduced by he mal
p ocessing o echnical lignosul ona e, which is based on he oxida ion and hyd oly ic des uc ion o
lignin-con aining aw ma e ial [
1
,
2
]. I can p omo e plan g ow h and he e o e i is widely used o
ag icul u al and ho icul u al pu poses [
2
–
8
]. I was ound ha i s applica ion can educe leaching o
ni ogen in o he soil solu ion [
2
], educe he oxic e ec o pes icides, imp o e he ni ogen balance o
soils, inc ease he basal espi a ion o he soil, and has a posi i e e ec on soil biological ac i i y [9].
Lignohuma e oge he wi h o he humic and humic-like subs ances has a complex con o ma ional
a angemen and can be pe cei ed as sup amolecula s uc u es, he unc ioning o which is de e mined
by he composi ion, size o molecula uni s, and weak in e molecula o ces [
10
]. A majo aspec o
he sup amolecula a angemen is i s s abiliza ion by weak dispe sion o ces, whe e hyd ophobic
and hyd ogen bonds a e esponsible o hei appa en la ge molecula size [
11
]. In solu ions, humic
and humic-like subs ances can be micelle-like, sup a-molecula assemblies o small en i ies and hei
seconda y s uc u e can change signi ican ly wi h changing concen a ion and pH [
12
–
21
]. In soils,
humic subs ances a e spa ially a anged in dec easing o de o pola i y. This means ha highly pola
sup amolecula subuni s shield less pola subuni s agains he ee soil solu ion and o m laye s o
dec easing pola i y [22].
Molecules 2020,25, 5831; doi:10.3390/molecules25245831 www.mdpi.com/jou nal/molecules
Molecules 2020,25, 5831 2 o 13
Ini ially, he e m “lignohuma e” was cha ac e ized by Sp inge [
23
] as he humic ma e ial
wi h lowe ipeness and ni ogen con en and a ela i ely high p opo ion o sul onic acidic g oups
(as dis inguished om he “ ue” soil humic ma e ial). Lignohuma e has s uc u e and p ope ies
simila o humic subs ances isola ed om na i e ma ices. Du ing i s p oduc ion, he lignosul ona e is
ans o med in o a humic-like ma e ial (by means o a highe empe a u e and p essu e). Chemical
changes ongoing in he ans o ma ion a e simila o he humi ica ion o plan li e in na u e [
2
].
The inal p oduc (lignohuma e) usually has a lowe ni ogen con en and highe con en o sul onic
acidic g oups as well as o he O-con aining g oups (hyd oxyl, ca bonyl, ca boxyl, e he and es e ones)
in compa ison wi h humic subs ances isola ed om pea , soils, and sedimen s. In compa ison wi h
na i e humic subs ances, he lignohuma e can be cha ac e ized by he p esence o simple s uc u al
componen s o wide molecula he e ogenei y, smalle molecula size and weigh [2,24].
P esen s udies o lignohuma e a e ocused mainly on i s ag icul u al and ho icul u al
applica ions [
2
–
8
]; he e o e, s udies dealing wi h i s s uc u al cha ac e iza ion and o he p ope ies
a e ela i ely sca ce. Some wo ks [
2
,
24
] used chemical (elemen al analysis) and spec al (UV/VIS,
FT-IR nad
13
C-NMR) me hods o he cha ac e iza ion o lignohuma e and i s compa ison wi h
humic ma e ial ex ac ed om di e en na u al ma ices. The di usion o lignohuma e h ough plan
cu icles was s udied in ou p e ious wo k [
8
]. The cu icle was placed be ween wo cu e es illed
by aga ose hyd ogels. The dono hyd ogel was en iched by lignohuma e, he accep o hyd ogels
was p epa ed wi hou i (i.e., wi h ze o ini ial concen a ion o lignohuma e), and he anspo o
lignohuma e h ough cu icle was moni o ed. This wo k is ocused on he in insic di usion in he
hyd ogels. The di usion couple was assembled by means o he dono and accep o hyd ogels, bu no
cu icle was placed be ween hem and bo h hyd ogels we e in immedia e con ac a he in e ace.
In conside a ion o he he e ogeneous s uc u e o lignohuma e [
1
,
2
,
24
] and unusual esul s ob ained
o anspo p ope ies o aga ose hyd ogels con aining humic subs ances [
25
], he di usion o
lignohuma e in aga ose hyd ogels should be in es iga ed in de ail. I he di usion is ealized in he
wo hyd ogels, he lignohuma e has a concen a ion g adien in bo h pa s (dono and accep o ).
In con as , he solu ion can be s i ed, he e o e i has no concen a ion g adien in he dono solu ion.
The e o e, he di usion o lignohuma e om i s aqueous solu ion in o aga ose hyd ogel was also
in es iga ed in his wo k.
2. Resul s and Discussion
The i s expe imen al ask was o de e mine he di usion coe icien o lignohuma e in he
aga ose hyd ogel on he basis o expe imen al da a ob ained o he di usion couple desc ibed
abo e. I he couple is composed o hyd ogels o he same na u e, di e ing only in he lignohuma e
con en , he di usion coe icien should be he same in bo h pa s o couple— he dono and accep o
hyd ogels—in condi ions in which he di usion coe icien in no dependen on he concen a ion o
di usingpa icles[
26
–
28
]. Ma hema icaldesc ip iono di usionin hedi usioncoupleis ela i elyeasy.
Be o e he s a o di usion, he dono hyd ogel has a cons an concen a ion o lignohuma e c
0
in he
whole cu e e olume, whe eas i s concen a ion in he accep o hyd ogel is ze o. I bo h pa s o he
di usion couple can be conside ed as semi-in ini e mediums (meaning ha he concen a ions on he
ou side bo de s o he couple a e cons an and do no change in ime du ing di usion), he solu ion o
second Fick law is [26–28]
cx, =
1
2c0e c x
2√Dh , (1)
whe e c
x,
is he concen a ion o lignohuma e a posi ion xin ime and D
h
is he e ec i e di usion
coe icien o lignohuma e in hyd ogel. I can be seen ha he concen a ion o he di used componen
on he in e ace (ci) is ime independen and equal o c0/2.
Molecules 2020,25, 5831 3 o 13
To al di usion lux m
which goes h ough he in e ace be ween dono and accep o hyd ogels
(x=0) in ime can be calcula ed as [26–28]
m =c0 Dh
π. (2)
Equa ion (2) assumes ha he e is no accumula ion o lignohuma e a he in e ace, i.e., he di usion
lux om he dono hyd ogel o he in e ace is equal o he di usion lux om he in e ace in o he
accep o hyd ogel.
In Figu e 1, he examples o expe imen al da a a e shown. As expec ed, he amoun o lignohuma e
in he dono hyd ogel dec eased g adually as i di used h ough he in e ace in o he accep o hyd ogel.
Ou esul s con i m ha he expe imen ally de e mined dependency o m
on
√
is linea up o a
c i ical ime, when he equi emen o semi-in ini e mediums is no ul illed. The e o e, only he da a
co esponding wi h he abo emen ioned condi ions a e i ed by Equa ion (2).
Molecules 2020, 25, x FOR PEER REVIEW 3 o 13
𝑚=𝑐


 . (2)
Equa ion (2) assumes ha he e is no accumula ion o lignohuma e a he in e ace, i.e., he
di usion lux om he dono hyd ogel o he in e ace is equal o he di usion lux om he in e ace
in o he accep o hyd ogel.
In Figu e 1, he examples o expe imen al da a a e shown. As expec ed, he amoun o
lignohuma e in he dono hyd ogel dec eased g adually as i di used h ough he in e ace in o he
accep o hyd ogel. Ou esul s con i m ha he expe imen ally de e mined dependency o m
on √𝑡
is linea up o a c i ical ime, when he equi emen o semi-in ini e mediums is no ul illed.
The e o e, only he da a co esponding wi h he abo emen ioned condi ions a e i ed by Equa ion
(2).
(a)
(b)
Figu e 1. The ime de elopmen o o al amoun s o lignohuma e in dono (blue) and accep o ( ed)
hyd ogels (a); expe imen al da a i ed by Equa ion (2) (b).
The di usion coe icien o lignohuma e in aga ose hyd ogel D
h
was calcula ed on he basis o
Figu e 1b and Equa ion (2). I s alue was de e mined as 1.46 × 10
−10
m
2
/s (±8.66 × 10
−12
m
2
/s), which
ag ees wi h alues de e mined o humic subs ances by some o he au ho s [29–33]. Co nel e al. [29]
s udied he di usion o humic acids ac iona ed by means o ul a il a ion. They de e mined he
di usion coe icien s be ween 3.8 × 10
−11
and 1.6 × 10
−10
m
2
/s dependen on pH, ionic s eng h,
empe a u e, and humic molecula weigh . The dependence o he di usion coe icien on molecula
weigh s (acco ding o he S okes-Eins ein equa ion) was weake in compa ison wi h he esul s
ob ained o syn he ic polyme s. This anomalous beha io o humic acids was in e p e ed o be an
a e ac o he de e mina ion o molecula weigh s by ul a il a ion. The au ho s concluded ha ue
molecula weigh s o humic ac ions we e subs an ially lowe han he nominal alues o
ul a il a ion. Pinhei o e al. [30] used ol amme ic echniques o he de e mina ion o di usion
coe icien s o humic and ul ic acids and hei me al complexes. They ob ained esul s in he ange
o om 5 × 10
−12
o 2 × 10
−11
m
2
/s o humic acids and hei complexes and om 6 × 10
−11
o 1.2 × 10
−12
m
2
/s o ul ic acids and hei complexes. Lead e al. [31] s udied he di usion o humic and ul ic
acids isola ed om he Suwannee i e (s anda d o In e na ional Humic Subs ances Socie y) by
means o he luo escence co ela ion spec oscopy. They de e mined he di usion coe icien s in he
ange o (2–3) × 10
−10
m
2
/s. They obse ed he agg ega ion o humic subs ances a lowe pH alues.
No e ec o concen a ion and ionic s eng h was obse ed. Lead and Wilkinson [32] used he same
me hod o he de e mina ion o di usion coe icien s o di e en humic subs ances in wa e and
aga ose hyd ogel. In gene al, hei esul s we e in he ange o (1.9–2.3) × 10
−10
m
2
/s o all samples
Figu e 1.
The ime de elopmen o o al amoun s o lignohuma e in dono (blue) and accep o ( ed)
hyd ogels (a); expe imen al da a i ed by Equa ion (2) (b).
The di usion coe icien o lignohuma e in aga ose hyd ogel D
h
was calcula ed on he basis o
Figu e 1b and Equa ion (2). I s alue was de e mined as 1.46
×
10
−10
m
2
/s (
±8.66 ×10−12 m2/s
),
which ag ees wi h alues de e mined o humic subs ances by some o he au ho s [
29
–
33
].
Co nel e al. [29]
s udied he di usion o humic acids ac iona ed by means o ul a il a ion.
They de e mined he di usion coe icien s be ween 3.8
×
10
−11
and 1.6
×
10
−10
m
2
/s dependen
on pH, ionic s eng h, empe a u e, and humic molecula weigh . The dependence o he di usion
coe icien on molecula weigh s (acco ding o he S okes-Eins ein equa ion) was weake in compa ison
wi h he esul s ob ained o syn he ic polyme s. This anomalous beha io o humic acids was
in e p e ed o be an a e ac o he de e mina ion o molecula weigh s by ul a il a ion. The au ho s
concluded ha ue molecula weigh s o humic ac ions we e subs an ially lowe han he nominal
alues o ul a il a ion. Pinhei o e al. [
30
] used ol amme ic echniques o he de e mina ion o
di usion coe icien s o humic and ul ic acids and hei me al complexes. They ob ained esul s in
he ange o om 5
×
10
−12
o 2
×
10
−11
m
2
/s o humic acids and hei complexes and om 6
×
10
−11
o
1.2 ×10−12 m2/s
o ul ic acids and hei complexes. Lead e al. [
31
] s udied he di usion o humic
and ul ic acids isola ed om he Suwannee i e (s anda d o In e na ional Humic Subs ances Socie y)
by means o he luo escence co ela ion spec oscopy. They de e mined he di usion coe icien s
in he ange o (2–3)
×
10
−10
m
2
/s. They obse ed he agg ega ion o humic subs ances a lowe pH
alues. No e ec o concen a ion and ionic s eng h was obse ed. Lead and Wilkinson [
32
] used he
Molecules 2020,25, 5831 4 o 13
same me hod o he de e mina ion o di usion coe icien s o di e en humic subs ances in wa e and
aga ose hyd ogel. In gene al, hei esul s we e in he ange o (1.9–2.3)
×
10
−10
m
2
/s o all samples
unde all condi ions. Di usion coe icien s de e mined o hyd ogel we e app oxima ely 10–20% lowe
in compa ison wi h wa e , mainly due o inc eased pa h leng hs and o uosi y. They assumed ha
la ge agg ega es o humic subs ances we e excluded om he hyd ogel. In hei subsequen wo k [
33
],
he esul s ob ained by means o he luo escence co ela ion spec oscopy and classical di usion cells
we e compa ed. Dec eases in di usion coe icien s obse ed o some humic samples in hyd ogel
we e oo la ge o be explained by he o uosi y and obs uc i e e ec s o he hyd ogel po e s uc u e.
Ve y hyd ophobic humic subs ances we e p obably p e en ed om pene a ing he hyd ogel because
o hei agg ega ion. They s a ed ha a po en ial complexa ion o humic subs ances wi h he hyd ogel
ibe s is no impo an and can be neglec ed. In con as , he di usion coe icien s o humic acids in
wa e de e mined in e . [
34
] we e much lowe . They a ied be ween 2.3
×
10
−12
and 8.6
×
10
−12
m
2
/s
in he case o alues measu ed by means o dynamic ligh sca e ing. The alues based on ol amme y
we e in he ange o 4.4 ×10−12 and 1.3 ×10−11 m2/s.
The alue o he di usion coe icien (de e mined in his wo k) was used o he calcula ion o he
di usion p o ile acco ding o Equa ion (1). A compa ison o expe imen al da a wi h he compu ed
cu e is shown in Figu e 2. I seems ha ou expe imen al da a a e in a good ag eemen wi h he
simple ma hema ical model and he concen a ion o lignohuma e a he in e ace be ween dono and
accep o hyd ogels (c
i
) can be conside ed as he hal c
0
. Howe e , an in e es ing phenomenon was
obse ed when he di usion was con inued o se e al days. We can see ha ha one poin close o he
in e ace om he dono pa is highe han he hal c
0
a e one day o di usion (Figu e 2), as well as
when he di usion was p olonged (Figu e 3a). A e se e al days, he concen a ion o lignohuma e on
he ou side bo de o he dono pa dec eased and he concen a ion close o he in e ace inc eased
(Figu e 3b). A concen a ion jump was obse ed a he in e ace o longe imes. Wha is he eason
o his? I is no easy o explain. One possibili y is a il a ion e ec . The po e size o aga ose hyd ogel
was de e mined by a simple spec opho ome ic me hod [
35
] in ou p e ious wo k [
36
]. Thei a e age
diame e was de e mined as 360 nm. The Z-a e age diame e o lignohuma e dec eased wi h he
inc easing concen a ion o i s solu ion and i s alue was ~37 nm o 0.1% w . con en (Figu e 4a).
This means ha he po e size exceeds he S okes hyd odynamic adius o lignohuma e by almos
en imes.
Molecules 2020, 25, x FOR PEER REVIEW 4 o 13
unde all condi ions. Di usion coe icien s de e mined o hyd ogel we e app oxima ely 10–20%
lowe in compa ison wi h wa e , mainly due o inc eased pa h leng hs and o uosi y. They assumed
ha la ge agg ega es o humic subs ances we e excluded om he hyd ogel. In hei subsequen wo k
[33], he esul s ob ained by means o he luo escence co ela ion spec oscopy and classical di usion
cells we e compa ed. Dec eases in di usion coe icien s obse ed o some humic samples in
hyd ogel we e oo la ge o be explained by he o uosi y and obs uc i e e ec s o he hyd ogel po e
s uc u e. Ve y hyd ophobic humic subs ances we e p obably p e en ed om pene a ing he
hyd ogel because o hei agg ega ion. They s a ed ha a po en ial complexa ion o humic subs ances
wi h he hyd ogel ibe s is no impo an and can be neglec ed. In con as , he di usion coe icien s
o humic acids in wa e de e mined in e . [34] we e much lowe . They a ied be ween 2.3 × 10
−12
and
8.6 × 10
−12
m
2
/s in he case o alues measu ed by means o dynamic ligh sca e ing. The alues based
on ol amme y we e in he ange o 4.4 × 10
−12
and 1.3 × 10
−11
m
2
/s.
The alue o he di usion coe icien (de e mined in his wo k) was used o he calcula ion o
he di usion p o ile acco ding o Equa ion (1). A compa ison o expe imen al da a wi h he compu ed
cu e is shown in Figu e 2. I seems ha ou expe imen al da a a e in a good ag eemen wi h he
simple ma hema ical model and he concen a ion o lignohuma e a he in e ace be ween dono and
accep o hyd ogels (c
i
) can be conside ed as he hal c
0
. Howe e , an in e es ing phenomenon was
obse ed when he di usion was con inued o se e al days. We can see ha ha one poin close o
he in e ace om he dono pa is highe han he hal c
0
a e one day o di usion (Figu e 2), as
well as when he di usion was p olonged (Figu e 3a). A e se e al days, he concen a ion o
lignohuma e on he ou side bo de o he dono pa dec eased and he concen a ion close o he
in e ace inc eased (Figu e 3b). A concen a ion jump was obse ed a he in e ace o longe imes.
Wha is he eason o his? I is no easy o explain. One possibili y is a il a ion e ec . The po e size
o aga ose hyd ogel was de e mined by a simple spec opho ome ic me hod [35] in ou p e ious
wo k [36]. Thei a e age diame e was de e mined as 360 nm. The Z-a e age diame e o
lignohuma e dec eased wi h he inc easing concen a ion o i s solu ion and i s alue was ~37 nm o
0.1% w . con en (Figu e 4a). This means ha he po e size exceeds he S okes hyd odynamic adius
o lignohuma e by almos en imes.
Figu e 2. Concen a ion p o ile o lignohuma e in aga ose hyd ogel a e one day. The blue poin s
belong o he dono hyd ogel (dis ance x < 0), and he ed poin s belong o he accep o hyd ogel
(dis ance x > 0).
Figu e 2.
Concen a ion p o ile o lignohuma e in aga ose hyd ogel a e one day. The blue poin s
belong o he dono hyd ogel (dis ance x<0), and he ed poin s belong o he accep o hyd ogel
(dis ance x>0).
Molecules 2020,25, 5831 5 o 13
1
Figu e 3
Figu e 3.
Concen a ion p o iles o lignohuma e in aga ose hyd ogel a e wo (
a
) and en (
b
) days.
The blue poin s belong o he dono hyd ogel (dis ance x<0), he ed poin s belong o he accep o
hyd ogel (dis ance x>0).
Molecules 2020, 25, x FOR PEER REVIEW 5 o 13
Figu e 3. Concen a ion p o iles o lignohuma e in aga ose hyd ogel a e wo (a) and en (b) days.
The blue poin s belong o he dono hyd ogel (dis ance x < 0), he ed poin s belong o he accep o
hyd ogel (dis ance x > 0).
(a)
(b)
Figu e 4. Z-a e age diame e and E
4
/E
6
a io (a); polydispe si y and ze a po en ial o lignohuma e in
he dependence on i s concen a ion in aqueous solu ion (b).
The polydispe si y o 0.1% w . lignohuma e con en was app oxima ely 0.5, ze a po en ial a ound
−40 mV. Mo e nega i e ze a po en ial indica ed a highe s abili y o pa icles o lignohuma e. Many
au ho s (e.g., [37–39]) used he a io o abso bance a 465 o 665 nm (E
4
/E
6
, so called index o
humi ica ion) as an indica o o he a e age molecula weigh and size and wi h he oxygen con en
Figu e 4.
Z-a e age diame e and E
4
/E
6
a io (
a
); polydispe si y and ze a po en ial o lignohuma e in
he dependence on i s concen a ion in aqueous solu ion (b).
The polydispe si y o 0.1% w . lignohuma e con en was app oxima ely 0.5, ze a po en ial
a ound
−
40 mV. Mo e nega i e ze a po en ial indica ed a highe s abili y o pa icles o lignohuma e.
Many au ho s (e.g., [
37
–
39
]) used he a io o abso bance a 465 o 665 nm (E
4
/E
6
, so called index o
humi ica ion) as an indica o o he a e age molecula weigh and size and wi h he oxygen con en o

Molecules 2020,25, 5831 6 o 13
humic ma e ials. The E
4
/E
6
a io is usually <5 o humic acids [
1
,
24
,
37
,
38
,
40
]. Howe e , i was ound
ha he alues o he humi ica ion index E
4
/E
6
is highe han 5 o he con en o lignohuma e abo e
0.01% w . (see Figu e 4a). I may be indica i e o he p esence o O-con aining unc ional g oups
(hyd oxyl, ca bonyl, ca boxyl, and es e g oups) and lowe molecula weigh .
As can be seen, he E
4
/E
6
a io co esponds wi h he Z-a e age size o lignohuma e, and he e o e
i may be conside ed as an indica o o i s molecula size du ing he di usion in he aga ose hyd ogel.
Howe e , he si ua ion is mo e complex. In Figu e 5, he in ensi y, olume, and numbe pa icle
size dis ibu ions o lignohuma e a e shown. While he in ensi y and olume size dis ibu ions we e
i-modal, hei numbe -based dis ibu ion was mono-modal because he numbe o big pa icles
occupying a la ge olume was e y low. Despi e he ac ha he majo i y o lignohuma e pa icles
a e smalle han he a e age po e size in he hyd ogel, some o hem can be limi ed in hei mo ion,
which can esul in hei highe concen a ion close o he in e ace. Some s udies showed ha he
a e age size diame e is no a sui able pa ame e o cha ac e izing humic subs ances and should be
used only condi ionally as he appa en mean pa icle size in compa ison wi h o he wo ks [
41
,
42
].
As can be seen in Figu es 4and 5, he ela i ely high polydispe si y and mul i-modal cha ac e o
pa icle size dis ibu ions can suppo his conclusion. This means ha he lignohuma e can con ain
mo e ac ions wi h di e en mobili ies. The knowledge o pa icle size dis ibu ion is hus e y
impo an . The colloidal s abili y can hus a ec hei molecula o ganiza ion and e-a angemen in
he s udied sys em.
Molecules 2020, 25, x FOR PEER REVIEW 6 o 13
o humic ma e ials. The E
4
/E
6
a io is usually <5 o humic acids [1,24,37,38,40]. Howe e , i was ound
ha he alues o he humi ica ion index E
4
/E
6
is highe han 5 o he con en o lignohuma e abo e
0.01% w . (see Figu e 4a). I may be indica i e o he p esence o O-con aining unc ional g oups
(hyd oxyl, ca bonyl, ca boxyl, and es e g oups) and lowe molecula weigh .
As can be seen, he E
4
/E
6
a io co esponds wi h he Z-a e age size o lignohuma e, and he e o e
i may be conside ed as an indica o o i s molecula size du ing he di usion in he aga ose hyd ogel.
Howe e , he si ua ion is mo e complex. In Figu e 5, he in ensi y, olume, and numbe pa icle size
dis ibu ions o lignohuma e a e shown. While he in ensi y and olume size dis ibu ions we e i-
modal, hei numbe -based dis ibu ion was mono-modal because he numbe o big pa icles
occupying a la ge olume was e y low. Despi e he ac ha he majo i y o lignohuma e pa icles
a e smalle han he a e age po e size in he hyd ogel, some o hem can be limi ed in hei mo ion,
which can esul in hei highe concen a ion close o he in e ace. Some s udies showed ha he
a e age size diame e is no a sui able pa ame e o cha ac e izing humic subs ances and should be
used only condi ionally as he appa en mean pa icle size in compa ison wi h o he wo ks [41,42].
As can be seen in Figu es 4 and 5, he ela i ely high polydispe si y and mul i-modal cha ac e o
pa icle size dis ibu ions can suppo his conclusion. This means ha he lignohuma e can con ain
mo e ac ions wi h di e en mobili ies. The knowledge o pa icle size dis ibu ion is hus e y
impo an . The colloidal s abili y can hus a ec hei molecula o ganiza ion and e-a angemen in
he s udied sys em.
(a) (b)
(c)
Figu e 5. In ensi y (a), olume (b) and numbe (c) pa icle size dis ibu ions o lignohuma e wi h
concen a ion 0.001% w . (blue), 0.01% w . (g een), 0.1% w . ( ed) and 1% w . (black).
Figu e 5.
In ensi y (
a
), olume (
b
) and numbe (
c
) pa icle size dis ibu ions o lignohuma e wi h
concen a ion 0.001% w . (blue), 0.01% w . (g een), 0.1% w . ( ed) and 1% w . (black).
Molecules 2020,25, 5831 7 o 13
In he case o globula pa icles, hei di usion coe icien dec eases wi h he squa e o diame e :
D=kBT/(6πη ). (3)
whe e Dis he di usion coe icien (equal o D
h
in hyd ogel and o D
s
in solu ion), k
B
is he
Bol zmann cons an , Tis he empe a u e,
η
is he iscosi y o he medium and is he diame e o he
di using pa icle [
26
,
27
,
36
]. The e-a angemen (e.g., agg ega ion and des uc ion o weak bonds in
sup amolecula s uc u e o lignohuma e) can esul in changes in he size o pa icles, hei mobili y
and he po en ial accessibili y o ac i e si es o o he cons i uen s in p esen na u al sys ems.
As can be seen in Figu e 6, he E
4
/E
6
a io o lignohuma e in he hyd ogel changes wi h he posi ion
in he di usion couple. The alues o abso bance in he accep o hyd ogel o dis ances >20 mm
we e oo low, and he e o e he E
4
/E
6
a io was no calcula ed o hese dis ances om he in e ace.
I we compa e Figu es 3and 6, we can see ha he inc ease in lignohuma e concen a ion in he dono
hyd ogel is connec ed wi h he dec ease in he E
4
/E
6
a io. This means ha bigge pa icles cumula ed
in on o he in e ace and can pass h ough i only wi h di icul y. Simul aneously, he dono hyd ogel
is dep i ed o smalle pa icles di used h ough he in e ace in o he accep o hyd ogel, which caused
he inc ease in he E
4
/E
6
a io pas he in e ace. Changes obse ed in pa icle size dis ibu ions du ing
he di usion can esul in changes in lignohuma e molecula o ganiza ion. Smalle pa icles a e lea ing
he dono hyd ogel and he bigge pa icles accumula e in on o he in e ace. I smalle pa icles
we e cons i uen s o he sup amolecula s uc u e o lignohuma e and disappea ed om he dono
hyd ogel, he emaining bigge pa icles accumula ed in a ela i ely hin laye e-a anged and a new
spa ial o ganiza ion o p esen pa icles can be es ablished. New bonds be ween pa icles can o m
and o he s a e des oyed. The sup amolecula s uc u e changes a e dependen on he amoun and
composi ion o he p esen pa icles.
2
Figu e 6
Figu e 6. E4/E6 a io in aga ose hyd ogel a e wo (a) and en (b) days. The blue poin s belong o he
dono hyd ogel (dis ance x<0), while he ed poin s belong o he accep o hyd ogel (dis ance x>0).
Molecules 2020,25, 5831 8 o 13
In o de o in es iga e he di usion o lignohuma e in aga ose hyd ogel in de ail and unde s and
he obse ed di e ences be ween he ma hema ical model o he di usion couple (Equa ion (1)) and
he ob ained expe imen al da a (Figu e 3), he di usion o lignohuma e om i s solu ion in o he
hyd ogel was s udied. As desc ibed abo e, he pa icle size dis ibu ion and he s abili y o molecula
o ganiza ion a e s ongly in luenced by he con en o lignohuma e in i s solu ion. The dec ease in
i s concen a ion is connec ed wi h he dec ease in he colloidal s abili y and inc ease in he a e age
diame e and polydispe si y. On he o he hand, he dec ease in lignohuma e con en in he solu ion
caused by i s di usion in o he hyd ogel is connec ed wi h he deple ion o smalle pa icles and
e-a angemen o he sup amolecula s uc u e o lignohuma e. As we can see in Figu e 7, he E
4
/E
6
a io dec eases wi h he con inuing di usion, which indica es he inc ease in he pa icle diame e .
The inc ease in pa icle size is connec ed wi h a as e mobili y o smalle pa icles and hei di usion
in o he hyd ogel as well as he e-o ganiza ion o he sup amolecula s uc u e o lignohuma e.
In con as , he E
4
/E
6
a io in he icini y o he in e ace in hyd ogel has a maximum. This means ha
he i s laye in he hyd ogel is g adually sa u a ed by smalle pa icles om he solu ion and hese
pa icles can lea e i and di use in o mo e dis an laye s o he hyd ogel. I seems ha pa icles o
lignohuma e in he hyd ogel a e bigge han i s pa icles dissol ed in solu ion (mainly a he s a o
he expe imen ). I is impossible o de e mine a pa icula size on he basis o he alue o E
4
/E
6
a io.
This a io shouldbe conside edas anindica o o he changesin pa iclesize, and he e o ei canindica e
such p ocesses as he o ma ion o agg ega es and disin eg a ion o bigge agglome a es. I is necessa y
o ake in o conside a ion many ac o s and ci cums ances. One o hese is he ac ha i is di icul o
compa e pa icle size in di e en mediums—in solu ion and in hyd ogel. The abso bance (used o
he calcula ion o E
4
/E
6
a io) mus be co ec ed acco ding o he e ec ha he abso bance o pu e
medium has sub ac ed om ha measu ed o lignohuma e in a gi en medium a a gi en wa eleng h.
Ou p e ious esul s [
14
,
25
] show ha he addi ion o humic subs ances in o aga ose hyd ogels can
a ec hei beha io mainly om he poin o iew o i s heological p ope ies. The hyd ogels en iched
by humic subs ances we e mo e liquid in compa ison wi h he pu e aga ose hyd ogel and had a lowe
abili y o esis mechanical s esses. The e o e, he backg ound co ec ion by means o he abso bance
o pu e aga ose hyd ogel can shi he alues o E
4
/E
6
a io a he in e ace be ween di e en mediums.
Molecules 2020, 25, x FOR PEER REVIEW 8 o 13
In o de o in es iga e he di usion o lignohuma e in aga ose hyd ogel in de ail and unde s and
he obse ed di e ences be ween he ma hema ical model o he di usion couple (Equa ion (1)) and
he ob ained expe imen al da a (Figu e 3), he di usion o lignohuma e om i s solu ion in o he
hyd ogel was s udied. As desc ibed abo e, he pa icle size dis ibu ion and he s abili y o molecula
o ganiza ion a e s ongly in luenced by he con en o lignohuma e in i s solu ion. The dec ease in i s
concen a ion is connec ed wi h he dec ease in he colloidal s abili y and inc ease in he a e age
diame e and polydispe si y. On he o he hand, he dec ease in lignohuma e con en in he solu ion
caused by i s di usion in o he hyd ogel is connec ed wi h he deple ion o smalle pa icles and e-
a angemen o he sup amolecula s uc u e o lignohuma e. As we can see in Figu e 7, he E
4
/E
6
a io dec eases wi h he con inuing di usion, which indica es he inc ease in he pa icle diame e .
The inc ease in pa icle size is connec ed wi h a as e mobili y o smalle pa icles and hei di usion
in o he hyd ogel as well as he e-o ganiza ion o he sup amolecula s uc u e o lignohuma e. In
con as , he E
4
/E
6
a io in he icini y o he in e ace in hyd ogel has a maximum. This means ha
he i s laye in he hyd ogel is g adually sa u a ed by smalle pa icles om he solu ion and hese
pa icles can lea e i and di use in o mo e dis an laye s o he hyd ogel. I seems ha pa icles o
lignohuma e in he hyd ogel a e bigge han i s pa icles dissol ed in solu ion (mainly a he s a o
he expe imen ). I is impossible o de e mine a pa icula size on he basis o he alue o E
4
/E
6
a io.
This a io should be conside ed as an indica o o he changes in pa icle size, and he e o e i can
indica e such p ocesses as he o ma ion o agg ega es and disin eg a ion o bigge agglome a es. I
is necessa y o ake in o conside a ion many ac o s and ci cums ances. One o hese is he ac ha i
is di icul o compa e pa icle size in di e en mediums—in solu ion and in hyd ogel. The
abso bance (used o he calcula ion o E
4
/E
6
a io) mus be co ec ed acco ding o he e ec ha he
abso bance o pu e medium has sub ac ed om ha measu ed o lignohuma e in a gi en medium
a a gi en wa eleng h. Ou p e ious esul s [14,25] show ha he addi ion o humic subs ances in o
aga ose hyd ogels can a ec hei beha io mainly om he poin o iew o i s heological
p ope ies. The hyd ogels en iched by humic subs ances we e mo e liquid in compa ison wi h he
pu e aga ose hyd ogel and had a lowe abili y o esis mechanical s esses. The e o e, he
backg ound co ec ion by means o he abso bance o pu e aga ose hyd ogel can shi he alues o
E
4
/E
6
a io a he in e ace be ween di e en mediums.
(a)
(b)
Figu e 7. E
4
/E
6
a io o lignuhuma e in i s solu ion du ing i s di usion in o he hyd ogel (a); ime
de elopmen o E
4
/E
6
a io o lignohuma e in he hyd ogel a a dis ance o 0.5 mm om he in e ace
(b).
On he o he hand, he beha io o lignohuma e in di e en mediums can di e . Al hough he
size o po es is su icien ly la ge in he compa ison wi h lignohuma e pa icles [36], he en i ons o
Figu e 7.
E
4
/E
6
a io o lignuhuma e in i s solu ion du ing i s di usion in o he hyd ogel (
a
);
ime de elopmen o E
4
/E
6
a io o lignohuma e in he hyd ogel a a dis ance o 0.5 mm om he
in e ace (b).
On he o he hand, he beha io o lignohuma e in di e en mediums can di e . Al hough he
size o po es is su icien ly la ge in he compa ison wi h lignohuma e pa icles [
36
], he en i ons o
Molecules 2020,25, 5831 9 o 13
lignohuma e can in luence i s molecula o ganiza ion. The seconda y s uc u e o lignohuma e is hus
sensi i e o i s concen a ion and he medium in which i is loca ed. The pa icle size dis ibu ion and
he E
4
/E
6
a io can change wi h he posi ion in hyd ogel as a esul o p oceeding di usion, di e en
mobili ies o pa icles, hei con en in a gi en locali y, e c. The e o e, he sup amolecula s uc u e o
lignohuma e can eac sensi i ely on ac ual changes in he lignohuma e a angemen and su ounding
medium, which can a ec hei mobili y and pe meabili y in o di e en ma e ials. As men ioned abo e,
he lignohuma e is equen ly used in ag icul u al and ho icul u al applica ions. Nowadays, olia
applica ions o e ilize s and bio-s imulan s complemen s anda d oo e iliza ion [
8
,
43
]. Di usion
p ocesses play an impo an ole in bo h me hods o e iliza ion because he applica ion o e ilize s
is connec ed wi h he pene a ion in o plan o gans such as lea es and oo s. The sup amolecula
s uc u e o lignohuma e and i s abili y ega ding s uc u al e-a angemen hus can in luence i s
mobili y and pe meabili y in o plan s.
3. Ma e ials and Me hods
3.1. Chemicals
Aga ose (AG; ou ine use class; CAS 9012-36-6) was pu chased om Sigma-Ald ich (S . Luis,
MO, USA). Lignohuma e A was kindly p o ided by Amag o (P ague, Czech Republic). I ep esen s
a comme cial mix u e o huma es and ul a es p epa ed by he mal con e sion o echnical
lignosul ona es unde s ic ly con olled condi ions [
1
,
2
,
44
]. I s main cha ac e is ics, such as elemen al
composi ion and s uc u al ea u es, can be ound in e s. [1,8,24].
The pa icle size dis ibu ion and ze a po en ial o lignohuma e in wa e (0.005–10 g
·
dm
−3
)
we e measu ed by means o a Ze asize Nano ZS wi h backsca e ing de ec ion (Mal e n Ins umen s L d.,
Wo ces e shi e, UK) The diame e o pa icles was calcula ed by means o he Z-a e age unc ion (based on
he S okes-Eins ein equa ion, assuming pa icles o be sphe ical) [29,40,45,46].
3.2. P epa a ion o Hyd ogels
The me hod o p epa a ion o hyd ogels was desc ibed in de ail in ou p e ious s udies [
8
,
14
,
25
,
36
].
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 lignohuma e, hen hea ed (80
◦
C)
and s i ed o ob ain a anspa en solu ion, and inally sonica ed o emo e gases. A e wa ds, he
solu ion was pou ed in o he PMMA spec opho ome ic cu e e (inne dimensions: 10
×
10
×
45 mm).
The dono hyd ogel o di usion couple was p epa ed om 1% w . AG solu ion con aining 0.1% w .
o lignohuma e. The accep o hyd ogel was p epa ed using 1% w . AG solu ion ( ee o lignohuma e).
The pu e aga ose hyd ogel p epa ed in he same way as he accep o one was also used o he di usion
expe imen s moni o ing he anspo o lignohuma e om i s aqueous solu ion (0.1% w .).
The size o po es in aga ose hyd ogel was de e mined by he spec opho ome ic me hod published
by Ayma d e al. [35]. The a e age diame e o po es was de e mined as 0.36 µm [36].
3.3. Di usion Expe imen s
Two ypes o di usion expe imen s we e ealized. The i s one was he di usion o lignohuma e
in he di usion couple o med by he dono and accep o hyd ogels, while he second one was he
di usion o lignohuma e om i s solu ion in o he hyd ogel.
3.3.1. Di usion Couple
The di usion couple was ealized by connec ing wo cu e es illed by aga ose hyd ogels, as
desc ibed in e . [
8
]. The dono hyd ogel con ained homogeneously dispe sed lignohuma e (0.1% w .),
and he accep o hyd ogel was p epa ed as a pu e one wi hou lignohuma e. Du ing he di usion
expe imen s, all he di usion couples we e placed in a closed con aine abo e wa e le el (in o de o
main ain cons an humidi y o he su oundings). Expe imen al condi ions ( ela i e humidi y 100%,