Ci a ion: Ma o , L.; Meile, K.;
Zoua i, M.; DeVallance, D.; Sandak,
A.; He e a, R. Cha ac e iza ion o
he Compounds Released in he
Gaseous Was e S eam du ing he
Slow Py olysis o Hemp (Cannabis
sa i a L.). Molecules 2022,27, 2794.
h ps://doi.o g/10.3390/
molecules27092794
Academic Edi o : Se e ina Paci ico
Recei ed: 4 Ap il 2022
Accep ed: 23 Ap il 2022
Published: 27 Ap il 2022
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molecules
A icle
Cha ac e iza ion o he Compounds Released in he Gaseous
Was e S eam du ing he Slow Py olysis o Hemp
(Cannabis sa i a L.)
Lae i ia Ma o 1,2 , K is ine Meile 3, Ma iem Zoua i 1,4 , Da id DeVallance 1,4, Anna Sandak 1,4
and Rene He e a 1,5,*
1InnoRenew CoE, Li ade 6, 6310 Izola, Slo enia; lae i ia.ma o @inno enew.eu (L.M.);
ma iem.zoua i@inno enew.eu (M.Z.); de allance@inno enew.eu (D.D.); anna.sandak@inno enew.eu (A.S.)
2And ej Ma ušiˇc Ins i u e, Uni e si y o P imo ska, Muzejski T g 2, 6000 Kope , Slo enia
3Bio e ine y Labo a o y, La ian S a e Ins i u e o Wood Chemis y, S . Dze benes 27, LV-1006 Riga, La ia;
[email p o ec ed]
4Facul y o Ma hema ics Na u al Sciences and In o ma ion Technologies, Uni e si y o P imo ska,
Muzejski T g 2, 6000 Kope , Slo enia
5Chemical and En i onmen al Enginee ing Depa men , Uni e si y o he Basque Coun y (UPV/EHU),
Plaza Eu opa 1, 20018 San Sebas ián, Spain
*Co espondence: enealexande [email p o ec ed] o ene.he diaz@inno enew.eu
Abs ac :
This s udy aims o cha ac e ize and alo ize hemp esidual biomass by a slow py olysis
p ocess. The ola ile by-p oduc s o hemp ca boniza ion we e cha ac e ized by se e al me hods (TGA,
UV-VIS, TLC, Flash P ep-LC, UHPLC, QTOF-MS) o unde s and he py olysis eac ion mechanisms
and o iden i y he chemical p oduc s p oduced du ing he p ocess. The ob ained ca bon yield was
29%, gene a ing a gaseous s eam composed o phenols and u ans which was collec ed in ou
empe a u e anges (F1 a 20–150
◦
C, F2 a 150–250
◦
C, F3 a 250–400
◦
C and F4 a 400–1000
◦
C).
The ob ained liquid ac ions we e sepa a ed in o sub ac ions by lash ch oma og aphy. The o al
phenolic con en (TPC) a ied depending on he ac ion bu did no co ela e wi h an inc ease
in empe a u e o wi h a dec ease in pH alue. Compounds p esen in ac ions F1, F3 and F4,
being mainly phenolic molecules such as guaiacyl o sy ingyl de i a i es issued om he lignin
deg ada ion, exhibi an ioxidan capaci y. The empe a u e o he py olysis p ocess was posi i ely
co ela ed wi h de ec able phenolic con en , which can be explained by he decomposi ion o de o
he hemp chemical cons i uen s. A de ailed unde s anding o he chemical composi ion o py olysis
p oduc s o hemp esiduals allows o an assessmen o hei po en ial alo iza ion ou es and he
u u e economic po en ial o unde u ilized biomass.
Keywo ds:
esidual s eam; hemp by-p oduc s; slow py olysis; gaseous phase; he mal decomposi-
ion; biocompounds
1. In oduc ion
Hemp is an annual c op cul i a ed wo ldwide o applica ions in a ious indus ies
such as ex ile and pape (hemp ib es), ood and eed (hemp seeds), cons uc ion (hemp
shi es), and medicine wi h he ex ac ion o cannabidiol (CBD) om he hemp lowe s
and lea es [
1
]. In Wes e n Eu opean coun ies, hemp is mainly cul i a ed o CBD p o-
duc ion, while he whole s em, including ib es and shi es, becomes a was e o low- alue
by-p oduc [
2
]. Unp ocessed hemp s em biomass is hen used as a e ilize o ene gy
sou ce, bu hese applica ions do no b ing much alue o he biomass, which could ins ead
be in eg a ed in o a alo iza ion chain and hus ul il he sus ainable ci cula economy
p inciples [
3
]. In e es in manu ac u ing wi h enewable ma e ials and compounds in-
s ead o ossil esou ces is ising due o he socie al demand o biobased p oduc s and
sus ainabili y awa eness.
Molecules 2022,27, 2794. h ps://doi.o g/10.3390/molecules27092794 h ps://www.mdpi.com/jou nal/molecules
Molecules 2022,27, 2794 2 o 13
A p omising solu ion o alo ize esidual hemp ma e ial is he mochemical con e sion
in o biocha (o bioca bon). Biocha is a ca bonaceous p oduc ob ained om biomass
eeds ocks hea ed a ele a ed empe a u es in an oxygen-s a ed a mosphe e [
4
]. Thanks
o i s in e es ing p ope ies, biocha has been in es iga ed o a wide ange o low o
high- alue applica ions such as soil e ilize [
5
], solid uel and elec ical componen s [
6
],
senso s [
7
], and componen s in ba e ies and supe capaci o s [
8
]. Biocha ’s physical and
chemical p ope ies can be adap ed by changing p oduc ion p ocess pa ame e s and by
choice o eeds ock. Biocha can be ob ained om se e al he mochemical ans o ma-
ions, including slow and as py olysis, gasi ica ion, hyd o he mal ca boniza ion, lash
ca boniza ion and o e ac ion [
9
]. These me hods di e mainly by inal empe a u es
and hea ing a es, which will de e mine he yield and quali y (e.g., su ace a ea and po e
olume) o biocha and, consequen ly, i s p ope ies and p ice. Slow py olysis, also called
con en ional ca boniza ion, and hyd o he mal ca boniza ion a e wo o he mos e icien
biocha con e sion echnologies which can be used o a wide ange o eeds ocks [
10
].
While he quali y o biocha gene ally inc eases wi h he empe a u e o he py olysis
p ocess, i s yield dec eases, and he yield o gases inc eases. Hence, he p oduc ion o
high-quali y biocha o en gene a es la ge amoun s o gaseous esidues.
Valo iza ion o hese esidues is needed o imp o e he e iciency o o e all biocha
p oduc ion [
11
]. Fo example, Xin e al. [
12
] s udied he economic ad an age o cop oducing
liquid smoke ood la ou ing along wi h bio uels wi hin a small-scale as py olysis p ocess.
Mo eo e , he alo iza ion o compounds om he mochemical con e sion was e s eams
allows o an inc ease in he p o i abili y o he p ocess. Ini ially, he deg ada ion o
hemicelluloses occu s, which leads o he o ma ion o u u als and ace ic acid. When
empe a u es inc ease, he lignin deg ada ion p ocess p oduces phenolic compounds
o di e en molecula weigh s, as well as acids, alcohols, aldehydes, ke one es e s and
he e ocyclic de i a i es. The complex aqueous s eams gene a ed om py olysis p ocesses
con ains solubilized o ganic compounds which could be collec ed and used o o he
applica ions, hus o e ing economical po en ial and p omo ing he ansi ion o a ming
owa ds ze o was e wi h a neu al o posi i e en i onmen al impac . Wi hou a alo iza ion
s a egy, he was e hen becomes an economic bu den o he mochemical bio e ine ies,
which need o ea and ecycle he e luen s. Li e a u e highligh s alo iza ion o se e al
indus ial sec o s, especially oods, cosme ics, and pha maceu ics [
13
]. Wilson e al. [
14
]
isola ed high pu i y phenol and ca echol om aqueous was e s eams gene a ed ia as
ca aly ic py olysis. Addi ionally, componen s om he was e s eams p esen ed in e es ing
p ope ies such as an ioxidan ac i i y, an imic obial, an i i al, o an i-alle genic e ec ,
ood addi i es and ag ances [
15
–
18
]. Salami e al. [
19
] in es iga ed he po en ial o using
slow py olysis o hemp shi es, oo s and lea es, and cha ac e ized he liquids e ie ed
when empe a u es we e be ween 275–350
◦
C. Thei esea ch ound ha he py olysis
p ocessing o hemp ma e ials esul ed in eco e ed ace ic acid, 2,6-dime hoxyphenol, 2-
me hoxyphenol, and cannabidiol liquids. In a simila s udy, he esul ing dis illa es o
h ee ypes o hemp shi es hea ed using slow py olysis up o 350
◦
C we e iden i ied
o p oduce baseline da a o po en ial hemp alo iza ion [
20
]. Ba be o-López e al. [
21
]
also in es iga ed dis illa es om hemp ib e py olysis and ound ha ace ic acid was he
main chemical cons i uen . Addi ionally, he dis illa es om hemp had highe amoun s o
me hanol, as compa ed o sp uce and bi ch py olyzed unde he same condi ions.
The objec i e o his s udy was o in es iga e he ola ile by-p oduc s o hemp ca -
boniza ion, wi h he pu pose o iden i ying he possibili ies o alo ize he chemical p od-
uc s which a ise du ing slow py olysis. Addi ionally, an analy ical app oach was imple-
men ed o be e unde s and he py olysis eac ion mechanisms. The componen s we e
ini ially sepa a ed in o ac ions acco ding o he ange o deg ada ion empe a u es. The
ac ions we e isola ed, and hei phenolic con en and po en ial an ioxidan ac i i y we e
explo ed and hen inally iden i ied h ough ch oma og aphy.
Molecules 2022,27, 2794 3 o 13
2. Resul s and Discussion
2.1. The mochemical Deg ada ion o Hemp
Figu e 1shows he he mog a ime ic deg ada ion o hemp om 0 o 800
◦
C unde
N
2
a mosphe e. Lignin is epo ed o slowly decompose o e a b oade empe a u e ange
han cellulose and he hemicellulose componen s o hemp [
22
], so pa o he weigh loss o
each empe a u e ange includes lignin- ela ed compounds. Be ween 20 and
150 ◦C
, an
endo he mic a ea appea s co esponding o mois u e and he delica e ola iles (ex ac i es)
eleased, eaching a 5.6% weigh loss wi h a hea ing a e o 1.67
◦
C/min. Be ween 150
and 250
◦
C, hemicelluloses s a o deg ade (8.5% ou o 24.1% o en-d ied basis epo ed
hemicellulose composi ion) [
22
–
25
], and be ween 250 and 400
◦
C, he majo mass loss
(54.6%) occu s which co esponds o he supe posi ion o he decomposi ion o he emain-
ing hemicelluloses (abou 15.6%), and he decomposi ion o cellulose (46.1% o en-d ied
basis epo ed cellulose composi ion). The maximum deg ada ion o he basic o ganic
componen s occu s be ween 315
◦
C and 400
◦
C [
23
–
26
], whe e an exo he mic peak om
depolyme iza ion o he hemicelluloses was obse ed a 320
◦
C. F om 400 o 800
◦
C, lignin
decomposi ion con inues (22.81% o en-d ied basis epo ed lignin composi ion), and he
sum o he mass losses achie ed a e decomposi ion co esponds o 77.20%.
Figu e 1. The mog a ime ic deg ada ion o hemp om 0 o 800 ◦C unde N2a mosphe e.
A e he mochemical con e sion (un il 1000
◦
C) in he ube u nace, 57.4 g o solid
esidue was ob ained, which esul ed in a 29.2% yield. In compa ison, a e TGA analysis
(up o 800
◦
C), less solid esidue was ob ained (22.2
±
1.8%). The di e ence be ween
he esidual con en om he mochemical con e sion and om TGA is mainly due o a
scale e ec and equipmen p ecision [
26
], he lowe scale o analysis in he TGA ( ew mg),
and highe weighing accu acy compa ed o he amoun con e ed in he ube u nace
could gene a e hese di e ences. Ne e heless, he in o ma ion p o ided by he TGA was
decisi e o he de ini ion o he collec ed anges based on he peaks and segmen s d awn
in he he mog a ime ic di e en ial.
2.2. Cha ac e iza ion o he Slow Py olysis Vola ile F ac ions
Based on he p e iously discussed TGA esul s, ola iles a ising in he slow py olysis
p ocess we e cap u ed a he exi o he ube u nace as ac ions a di e en empe a u es:
F1 a 20–150
◦
C, F2 a 150–250
◦
C, F3 a 250–400
◦
C, and F4 a 400–800
◦
C. The collec ed
ac ions unde wen a se ies o cha ac e iza ions, and he esul s a e summed up in Table 1.
The concen a ion o he non- ola ile compounds in he ac ion up o 150
◦
C (F1) was
6–8 imes
lowe han in he subsequen ac ions (F2–F4), whose concen a ions we e ela-
i ely simila . These alues can be co ela ed wi h he weigh loss ound in he TGA, whe e
Molecules 2022,27, 2794 4 o 13
he la ges mass loss was ound in F3, while F2 and F4 p esen alues wi hin he same ange.
In addi ion, he acid numbe (AN) was e alua ed as a p edic ion alue o p oduc s p esen
in he ac ions, such as phenolic acids and ligh oxygena ed o ganic compounds. Vola ile
acids, mainly ace ic acid, a e conside ed o be po en ial p oduc s ha can be eco e ed
om py olysis p oduc s [
25
]. The AN inc eased du ing he he mochemical con e sion,
ob aining he highes alues om 400–800
◦
C (F4). Howe e , since he compounds p esen
in he ac ions we e dilu ed wi h he cap u ing wa e , he ANs in all he ac ions we e low
(<2) compa ed wi h ha ound in he li e a u e [
24
,
26
]. Mo eo e , he pH ange a ied om
5.5 o 3.5, which indica es ha he concen a ion o weak o ganic acids inc eased du ing
he he mochemical con e sion o hemp.
Table 1. Gene al analysis esul s o he slow py olysis ola ile p oduc ac ions.
Pa ame e F1 F2 F3 F4
Tempe a u e ange 20–150 ◦C 150–250 ◦C 250–400 ◦C 400–800 ◦C
pH 5.48 4.59 3.47 3.72
Acid numbe , mg KOH/g 0.136 0.364 0.815 1.148
Non- ola ile concen a ion, mg/g 0.041 0.277 0.344 0.244
TPC, µg GAE/mg sample 1315.00 65.68 1027.99 1833.08
IC50 (DPPH), ug/mL 2.951 40.593 3.155 2.121
Wi h espec o he o al phenolic con en (TPC) calcula ed on each ac ion, he o de
was as ollows: F4 > F1 > F3 > F2. These esul s showed ha TPC a ied depending on
he ac ion bu did no co ela e wi h he inc ease in empe a u e o wi h he dec ease
o he pH alue, implying ha he acidi y o he collec ed ac ions was mo e ela ed o
low molecula ola ile acids a he han o phenol ca boxylic acids. In gene al, he ac ion
ha is composed mainly o lignin deg ada ion p oduc s (F4) showed he highes TPC,
while he ac ion including he main deg ada ion p oduc s om hemicelluloses (F2) had
he lowes TPC. Mo eo e , he mos ola ile ac ion (F1) showed high TPC, al hough he
g a ime ic concen a ion was qui e low. The lack o co ela ion be ween he g a ime ically
de e mined non- ola iles and he chemically-de e mined phenols was ela ed o phenol loss
while d ying a 105
◦
C. These esul s ag ee wi h hose ound in p e ious s udies in which
signi ican concen a ions o phenol compounds we e obse ed in he ola ile ac ions
o hemp as py olysis (up o 560
◦
C) [
27
] and in he hemp slow py olysis dis illa es [
20
].
The main cons i uen s ound include monophenols, phenol de i a i es, guaiacols and
sy ingols [28,29].
The an ioxidan ac i i y o he ac ions was de e mined by he DPPH assay (IC50),
in which he mos ac i e ac ions need a lowe concen a ion o inhibi 50% o he DPPH
adical used in he e alua ion. A linea and posi i e co ela ion was obse ed be ween
TPC and DPPH con en (R = 0.73), whe e he ac ions wi h high TPC con ibu ed signi i-
can ly o he DPPH adical sca enging capaci y o he ac ion. These p elimina y esul s
showed ha compounds p esen in ac ions F1, F3, and F4 po en ially ac as an ioxidan s,
being mainly phenolic molecules such as guaiacyl o sy ingyl de i a i es issued om he
lignin deg ada ion [
14
,
15
,
30
–
32
]; howe e , u ans a e also epo ed o show an ioxidan
p ope ies [15].
Mo eo e , UHPLC analysis was pe o med o acqui e mo e de ailed in o ma ion abou
he chemical composi ion o he ac ions. Figu e 2shows he UHPLC-UV
(λ= 280 nm
)
ch oma og ams o he ac ions. This analy ical me hod is aimed a de e mining u an
and phenol de i a i es bu excludes low molecula weigh p oduc s, such as o mic acid,
o maldehyde, ace aldehyde, e c. Based on he e en ion imes o he s anda ds, an app oxi-
ma e di ision be ween u ans and phenols was made a 4 min (Figu e 2). Table 2shows he
ela i e quan i a i e compa ison o hese ac ions based on he o al peak a ea up o
4 min
and a e 4 min a 280 nm wa eleng h. The summed peak a eas o u ans and phenols
showed ha ac ions F1 and F2 had a e y low concen a ion o cons i uen s (2.2% o he
o al de ec ed analy es) as compa ed o F3 and F4, meaning ha he yield o condensable
u an and phenol p oduc s a py olysis empe a u es up o 250 ◦C was almos negligible.
Molecules 2022,27, 2794 5 o 13
Figu e 2.
UHPLV-UV ch oma og ams (
λ
= 280 nm) o he main ac ions o py olysis was e s eams.
Table 2. UHPLC-UV analysis o he slow py olysis ola ile p oduc ac ions.
Compounds F1 F2 F3 F4
5-HMF, µg/g 0.13 0.42 1.2 n
Fu u al, µg/g 0.74 0.14 32 15
Fu an (1.87 min *), el. uni s 0.14 0.047 0.24 0.23
Fu ans (<4 min *), el. uni s 0.31 0.30 7.0 5.0
Phenols (>4 min *), el. uni s 0.12 0.12 2.1 4.4
Phenol (4.12 min *), el. uni s n n 0.40 1.0
Phenol (4.42 min *), el. uni s 0.05 n 0.03 0.05
Phenol (4.49 min *), el. uni s n n 0.20 0.34
Phenol (4.54 min *), el. uni s n n n 0.17
Phenol (4.93 min *) el. uni s n n 0.17 0.28
Phenol (5.45 min *), el. uni s n n n 0.19
Phenol (5.55 min *) el. uni s n n n 0.090
* Re en ion ime in UHPLC-UV ch oma og ams (Figu e 2).
The yield o condensable u an and phenol p oduc s a highe empe a u es was
simila ly dis ibu ed be ween ac ions F3 (47.3%) and F4 (48.3%). Howe e , wi h inc easing
empe a u e, he a io be ween u ans and phenols changed. Namely, he highe he
py olysis empe a u e, he mo e phenols could be de ec ed in he sample. This is explained
by he mo e easily-occu ing he mal decomposi ion o holocellulose (sou ce o u ans),
ollowed by he decomposi ion o lignin (sou ce o phenols) wi h mo e se e e py olysis
ea men . By compa ison o s anda ds, peaks wi h
R
2.22 and 3.28 min we e iden i ied as
5-HMF and u u al, espec i ely. Fu u al was he dominan u an in all ac ions. The
ela i e concen a ion o 5-HMF was signi ican ly lowe and below he de ec ion limi in
F4. Concen a ion o 5-HMF in F1, F2, and F3 was 0.13, 0.42, and 1.2
µ
g/mL, espec i ely.
Fu u al was 0.74, 0.14, 32, and 15
µ
g/mL in F1, F2, F3, and F4, espec i ely. Fu u al
has been desc ibed as a aluable pla o m chemical ob ainable by py olysis om di e en
biomass eeds ocks [30].
2.3. Sepa a ion o he F ac ions in o Sub ac ions
To expand he cha ac e iza ion and o explo e he possibili ies o speci ic chemical
compounds p oduc ion, he ac ions collec ed om he ube u nace we e u he sepa a ed
by p epa a i e liquid ch oma og aphy. Fo each ac ion, he choice o he mos sui able
eluen was de ined hanks o he hin laye ch oma og aphy (TLC) sc eening. The mos
e ec i e mobile phase obse ed wi h TLC was ace one–e hanol; hus, a s epwise g adien
was op imized o he ac iona ion p ocess om 100:0 o 90:10 ( / ). A mix u e o ace one
(sol en A) and e hanol (sol en B) wi h an inc easing g adien a io was used o sepa a e
each ac ion in o sub ac ions (S ) in he p epa a i e ch oma og aphy sys em, based on
hei a ini y wi h he eluen and on hei UV and ELS signals.
Molecules 2022,27, 2794 6 o 13
Table 3shows he esul s o he ac iona ion acco ding o he ELSD and UV abso bance
o he whole UV spec a and o some speci ic wa eleng hs (254, 265, 280 and 320 nm).
The chemical compounds con ained in each ac ion ha e di e en UV spec a, and hus a
di e en esponse ac o a a speci ic wa eleng h. Howe e , i was obse ed ha he sample
esponse co esponding o he wa eleng h a 280 nm was he closes o he esponse o he
whole UV spec a. Based on hese esul s, F1 was decomposed in o ou sub ac ions (S -1
o S -4), in which S -1 and S -4 con ain only UV-de ec ed g oups, and S -2 and S -3 con ain
compounds de ec ed by ELSD. F2 was decomposed in o h ee sub ac ions, all o hem
sepa a ed exclusi ely by UV signals. Mo eo e , F3 was di ided in o h ee sub ac ions
whe e S -1 con ains only UV-de ec ed g oups, S -2 con ains ELSD g oups and S -3 is a
mix u e o compounds de ec ed wi h bo h UV and ELSD de ec o s. F ac ion 4 was di ided
in o h ee sub ac ions whe e S -1 and S -3 con ain UV de ec ed g oups and S -2 p esen s
only compounds wi h ELSD ac i i y.
Table 3. Desc ip ion o he sub ac ions ob ained by p epa a i e ch oma og aphy.
Sub ac ions/F ac ions S -1 S -2 S -3 S -4
Sol en a io (A:B) 100:0 →99:1 99:1 →98:2 98:2 →97:3 97:3 →90:10
F1 Con ains 1 g oup
(UV-de ec ed)
Con ains mix u e g oups
(ELSD-UV-de ec ed)
Con ains mix u e g oups
(ELSD-UV-de ec ed)
Con ains 1 g oup
(UV-de ec ed)
F2 Con ains 1 g oup
(UV-de ec ed)
Con ains 1 g oup
(UV-de ec ed)
Con ains 1 g oup
(UV-de ec ed) -
F3 Con ains 1 g oup
(UV-de ec ed)
Con ains 1 g oup
(ELSD-de ec ed)
Con ains mix u e g oups
(ELSD-UV-de ec ed) -
F4 Con ains 1 g oup
(UV-de ec ed)
Con ains 1 g oup
(ELSD-de ec ed)
Con ains 1 g oup
(UV-de ec ed) -
2.4. Quali a i e Cha ac e iza ion o he F ac ions and Sub ac ions
Quali a i e analysis o he ac ions and sub ac ions was comple ed by he mo e
sensi i e QTOF-MS de ec ion. In LC-MS, he esponse o a chemical is in luenced by
he chemical’s s uc u e and chemical quali ies and also i s in e ac ion wi h he di e en
pa ame e s o he MS ins umen . We obse ed ha in he gi en condi ions used o
un a ge ed MS analysis, some analy es did no ionize a all ( u u al), bu some p oduced
ions ha did no ma ch hei molecula ions (5-HMF). Howe e , many a oma ic s uc u es
we e de ec ed and iden i ied.
The o mulas o he main s uc u es de ec ed in he ac ions F1, F2, F3, and F4 can
be cau iously iden i ied as ollows. The peak wi h
R
1.87 min had he mos in ensi e
ion
125.03 Da
, co esponding o C
6
H
6
O
3
, he molecula s uc u e o me hyl u oic acid.
Peak wi h
R
4.12 min and 139.08 Da ion was ela ed o he s uc u e C
7
H
8
O
3
. Peak wi h
R4.42 min
and 123.05 Da ion i s wi h C
7
H
8
O
2
me hoxyphenol (guaiacyl) s uc u e. A
4.49 min
he e was also a s uc u e wi h a 123.05 Da ion, a agmen o a guaiacol de i a i e.
The o he peaks, e en hough adequa ely sepa a ed by UV de ec ion, we e no esol ed
in he MS ch oma og ams, because e en low concen a ions o co-elu ing compounds
in e e ed wi h he iden i ica ion o peaks a e 4.5 min. Namely, he e we e mo e de ec ed
peaks wi h MS (Figu e 3) han wi h UV de ec ion, making i di icul o assign a mass
spec um o a peak in he UV ch oma og am.
The main iden i ied s uc u es, ei he molecula o agmen ions, a e shown in
Figu e 4. The peaks wi h ea lie elu ion and ions wi h mass 125.03 and 109.03 Da we e
con i med o be u an de i a i es—me hyl- u oic acid and ace yl u an. Peaks wi h la e elu-
ion we e phenols wi h ypical s uc u es, such as me hoxyphenol o guaiacol
(123.05 Da
),
dihyd oxybenzaldehyde (137.03), dime hoxybenzoic acid (181.04 Da) o o he isome s.
Figu e 3shows ha F3 and F4 we e quali a i ely e y simila . In F1 he e was a di e en
a io be ween u ans a and b and se e al phenols (peaks d,e,g). These la ge di e ences
we e no e iden in F3 and F4.
Molecules 2022,27, 2794 7 o 13
Figu e 3.
UHPLC-MS ch oma og ams o he was e s eam ac ions (
A
–
D
) and hei sub ac ions.
Iden i ied s uc u es in each peak: a—C
6
H
10
O
4
; b—C
6
H
6
O
3
; c—C
6
H
6
O
2
;
d—C7H6O3; e—C9H8O3
;
—C
8
H
8
O
3
; g—C
9
H
8
O
3
; h—C
9
H
10
O
4
; i—C
7
H
6
O
2
; j—C
7
H
8
O
2
; k—C
8
H
8
O
2
;
l—C8H10O2;
m—C10H10O3.
Molecules 2022,27, 2794 8 o 13
Figu e 4. Some o he chemical s uc u es iden i ied om he MS spec a (isome s possible).
F1 had a la ge numbe o indi idual compounds de ec ed bo h by UV and MS in
nega i e elec osp ay mode. Figu e 3A shows he UHPLC-MS ch oma og ams o sample F1
and i s sub ac ions. I appea s ha he sub ac ions S -1 and S -2 had a simila quali a i e
composi ion as he mo he ac ion. The main iden i ied chemical o mulas in F1 we e as
ollows: C
6
H
6
O
3
(125.03 Da), C
6
H
6
O
2
(109.03 Da), C
7
H
6
O
3
(137.03 Da), C
9
H
8
O
3
(163.04 Da),
C
7
H
8
O
2
(123.05 Da), C
10
H
10
O
4
(193.05 Da) and C
8
H
8
O
2
(135.05 Da). Fewe compounds
we e de ec ed in F2 (Figu e 3B).
The ac ions F3 and F4 (Figu e 3C,D) had e y simila quali a i e composi ions, wi h
quan i a i e di e ences, mos ly ega ding he a io be ween u ans and phenols. The main
s uc u es iden i ied we e C
6
H
6
O
3
(125.03 Da), C
6
H
6
O
2
(109.03 Da), C
8
H
8
O
3
(151.04 Da),
C
9
H
10
O
4
(181.04 Da), C
7
H
8
O
2
(123.05 Da), C
10
H
12
O
4
(195.07 Da), C
11
H
14
O
4
(209.09 Da),
C8H10O2(137.07 Da).
The p epa a i e HPLC sepa a ion o he F3 main ac ion had esul s simila o he
F1 sepa a ion—equal dis ibu ion be ween sub ac ions S -2 and S -4. Sepa a ion o he
F4 main ac ion esul ed in he sub ac ion S -2 wi h a simila quali a i e composi ion as
he main ac ion, while S -3 had only a ew chemical cons i uen s ela ed o he ollowing
s uc u es: C6H6O2(109.03 Da), C7H8O2(123.05 Da) and C8H10O2(137.07 Da).
3. Ma e ials and Me hods
3.1. Hemp
Hemp (Cannabis sa i a L.) om he Fu u a 75 a ie y was g own in 2020 in F ankolo o
(Slo enia) and supplied by he V hi šek a m. The composi ion o he hemp s ems used
in his s udy was p e iously assessed [
6
] as ollows:
α
-cellulose (46.09%), hemi-cellulose
(24.12%), Klason lignin (22.81%), o al ex ac i e compounds (4.14%) and ash (2.72%), on
an o en-d ied basis. The s ems we e s o ed in a d y en i onmen p io o unde going
he mochemical con e sion.
3.2. The mog a ime ic Analysis o Raw Hemp
The mog a ime ic analysis (TGA) was pe o med on 2–5 millig ams o hemp s em
wi h a Wa e s TA Ins umen TGA 5500 The mog a ime ic Analyze o isualize he
deg ada ion ange acco ding o he empe a u e o ea men . The samples we e hea ed
unde an ine a mosphe e (N
2
low 25 mL/min) om 20
◦
C o 400
◦
C a 1.67
◦
C/min,
and om 400
◦
C o 800
◦
C a 10
◦
C/min. The hea ing a es we e chosen o simula e he
condi ions o he he mochemical con e sion occu ing in he ube u nace, iden i ying he
key deg ada ion s eps o he p ocess and selec ing he ac ions o collec acco ding o he
deg ada ion o p oduc s om hemp [
6
]. Howe e , while he he mochemical con e sion in
Molecules 2022,27, 2794 9 o 13
he ube u nace was con inued up o 1000
◦
C, he TGA maximum empe a u e was limi ed
o 800 ◦C so as no o damage he pla inum pans con aining he samples.
3.3. The mochemical Con e sion
Hemp s ems (196.5 g) we e cu o smalle pieces o 15 cm leng h and placed in
ec angula alumina boa s inside a Nabe he m RSRC 120-1000/13 ube u nace. No d ying
s ep was added p io o he he mochemical con e sion in o de o p ese e he sensi i e
ola iles in he s ems. The he mochemical p ocess was pe o med unde an ine N
2
a mosphe e om 20 o 400
◦
C a 100
◦
C/h, and om 400
◦
C o 1000
◦
C a 600
◦
C/h. A
highe hea ing a e was chosen o he second pa o he con e sion o speed up he
p ocess, aking in o accoun ha mos o he ola iles a e eleased a empe a u es below
400
◦
C [
6
]. While he solid biocha esidue emains in he c ucibles, he ni ogen low
ca ies he gaseous compounds ou o he ube u nace h ough a pipe ending in a 500 mL
dis illed wa e ap (Figu e 5). A e he he mochemical con e sion cycle, he u nace was
cooled, and he solid esidues we e emo ed. The solid biocha esidue was weighed o
calcula e he yield based on he ollowing o mula (1).
Yield solid esidue (%)=Mass bioca bon (g)
Mass hemp biomass (g)×100 % (1)
Figu e 5.
Schema ic o he he mochemical con e sion p ocess and sampling o he aqueous ac ions.
3.4. Collec ion o he Was e S eams and Cha ac e iza ion
Fou empe a u e anges (20–150
◦
C, 150–250
◦
C, 250–400
◦
C and 400–1000
◦
C) we e
selec ed acco ding o he deg ada ion ange o he hemp mac o componen s, o collec
ac ions con aining he gaseous p oduc s apped in he wa e . A he end o each empe -
a u e ange, he inal empe a u e was main ained o 10 min o gi e ime o he sys em o
e acua e he gas p oduced du ing he co esponding s ep. A e each empe a u e ange,
he 500 mL o wa e was collec ed and eplaced wi h 500 mL o esh dis illed wa e o he
ollowing s ep.
The non- ola ile concen a ion (mg/g) o each ac ion was e alua ed by measu ing
10 mL in pe i dishes, placing hem a 105
◦
C and weighing he esidue. The acid numbe
o each ac ion was calcula ed by i a ion (mg KOH/g), and he pH was measu ed wi h
an elec ochemis y me e [The mo Scien i ic] (Wal ham, MA, USA).
3.5. To al Phenolic Con en and An ioxidan Ac i i y o he F ac ions
The o al phenolic con en o each ac ion was measu ed ollowing he Folin–Ciocal eu
me hod [
31
,
32
]. Fi s , se ial dilu ions o gallic acid solu ions in dis illed wa e we e p epa ed
and used la e o d aw a calib a ion cu e. Then, 0.3 mL o he solu ion o be es ed (each
