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1
Urea Enhanced NaOH Solvent and H2SO4
Enhanced DMSO Solvent System in The Study of Humic Substances
Department of Chem. & Environ. Sciences
[Guixue Song]
3
Soil Crude Humin
NaOH + 6 M Urea
Centrifuge SolubleInsoluble
HCl (pH 1 to 1.5)
Centrifuge
Insoluble Soluble
Urea HA Urea FA
Centrifuge
Insoluble
Insoluble
DMSO insoluble humin
DMSO humin
Dry samples DMSO + 6% H2SO4
H2O (pH 2)
Soluble
DMSO FA
10% HF
Comprehensive novel procedures for isolation of humin
4
Why urea?• Very soluble in water; a proton acceptor,
• Strong dipole moment (4.56)
• Good hydrogen bonder breaker in the biochemistry ( up to 9.5 M)
• Advantages: (1), high yield from 0.1 M NaOH + 6 M urea solvent, between 1 to 20% (by mass).
(2), water soluble. Urea residue can be easily washed out by acid wash and desalting steps in the XAD resin procedures.
(3), significant structural aspects changes were not observed.
5
• In the classical definitions the material isolated in the 0.1M NaOH + 6M urea system described would be regarded as Humin.
• Any material that can be considered to be humic that is extracted in organic solvents, after exhaustive extractions in base, would, in the classical definitions, be HUMIN
6
•Disadvantages: (1), urea could bond to humic molecules, or be trapped in humic matrix. Difficult to remove all the urea residues.
(2), possible oxidation (O2 uptaking) in high pH values of aqueous solution (> pH 12.6)
(3), possible side reactions by ureahydrolysis (release of ammonia) and functional groups of HS.
7
Develops odor
of ammonia4.56
26.9
(in DMSO)13.820.18
108 g/100 ml
(20 oC)1.33
OdorDipole
momentpK
apK
b
(H+-form)
pKa
Water
SolubilityDensity
132-15 oCDecompose07.2 (10%)
White
crystals
or white
powder
(NH2)2CO60.06
Melting PointBoiling Point% Volatile
(21 oC)
pH
(water)Appearance
Chemical
FormulaMW
Physical and chemical properties of urea
8
O
OHR
+NH3
O
NH2R
+ H2OH2
Note: the resonances of RCONH2 could be marked with other
amides in the resonances of 160 to 180 ppm in 13C NMR spectra.
H H
HH
Hydrogen bonding of urea and the carboxylate of HS
9
Why DMSO (dimethylsulfoxide)?• a highly polar, aprotic, water miscible organic solvent.
• Strong dipole moment of S=O bond (4.3), high dielectric constant (relative permittivity) (46.6).
• A powerful solvent (dissolve most aromatic and unsaturated hydrocarbons, organic N compounds, and many inorganic salts.
• Good solvent for cations and poor for anions. The exchangeable metal cations that neutralize the charges on the humic molecules in the divalent/polyvalent cation-exchanged soils would be solvated by DMSO.
• A good penetrating reagent for clay minerals
• A good solvent when mixed with small amount of acid (HCl or H2SO4)
10
• The oxygen of DMSO is somewhat basic and participates strongly as a hydrogen bond acceptor. A hypothetical representation of association between DMSO and the carboxyl, phenolic, and hydroxyl functional groups in HS,
(Ø-OH represents phenolic constituents and R represents the remainder of the
humic molecule.) (from Clapp et al, 2005).
11
Essentially odorless454.32.53Miscible1.10 g
per cm3
OdorDielectric
constant
Dipole
momentpK
HB
Water
SolubilityDensity
8518.5189 oC1.996colorless
liquid(CH
3)2S=O78.13
Flash pointMelting
Point oC
Boiling
Point oC
Viscosity
(20 oC)Appearance
Chemical
FormulaMW
Physical and chemical properties of DMSO
12
Schematics of the structure of the kaolinite/DMSO intercalation compound (after Hayashi, 1995). H: darkly and lightly shaded circles
Increase d (001)From 7.14 A to 11.16 A
13
NaOH plus urea solvent
Urea concentration (M)
Ab
sorb
an
ce (
40
0 n
m)
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
0 1 2 3 4 5 6 7 8 9
Urea+0.1 M NaOH, 1 h
Urea, 48 h
Urea + 0.1 M NaOH, 48 hA
bsorb
ance (
40
0 n
m)
Urea concentration (M)
14
NaOH plus urea solvent
0.1 M NaOH 0.1 M NaOH + 6 urea
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
0.1 M NaOH 0.1 M NaOH + 6 M urea
Ab
sorb
ance (
40
0 n
m)
Note, accurately weighted 1.000 g IHSS Mollisol soil, which had been exhaustively extracted with 0.1 M NaOH and dialysis to remove urea and soluble ions was used; the soil and solvent ratio was 1:20 (mass:volume).
Fu
lvic
fra
ction
15
0.2
0.21
0.22
0.23
0.24
0.25
0.26
0.27
0.28
0.29
0.3
H2SO4 HCL
Abs 4
00nm
Note: DMSO + 6% concd HCl or H2SO2 (v/v) solvent (10 ml) was mixed with dry (200 mg of the IHSS Mollisol standard) humin-enriched fine clay. DMSO extracts were diluted 20 times with distilled water, and left stand for overnight. The DMSO supernatant was used for absorbance measurement.
DMSO plus acid solvent
Ab
sorb
ance (
40
0 n
m)
DMSO + 6% (v/v) H2SO4 DMSO + 6% (v/v) HCl
Fu
lvic
fra
ction
16
14 13 12 11 10 9 8 7 6 5 4 3 2 1 ppm
A
B
C
D
E
x2~11% TOC
~9% TOC
~23% TOC
~24% TOC
~30% TOC
*
*
*
* §
§
§
§
*
123
x10
x10
14 13 12 11 10 9 8 7 6 5 4 3 2 1 ppm14 13 12 11 10 9 8 7 6 5 4 3 2 1 ppm
A
B
C
D
E
A
B
C
D
E
x2~11% TOC
~9% TOC
~23% TOC
~24% TOC
~30% TOC
*
*
*
* §
§
§
§
§
§
§
§
*
123 123
x10x10
x10x10
0.1 M NaOD
DMSO-d6
2.5% TFA in DMSO-d6
10% TFA in DMSO-d6
2.5% HSO4 in DMSO-d6
1= aliphatic species, 2 = predominantly carbohydrates, and 3 = aromatic and amide species. *protonated signals because of acids. § denotes DMSO signals
1H-NMR spectra of extracts from the soil (from Buum & Simpson, 2006)
17
12 11 10 9 8 7 6 5 4 3 2 1 ppm
A
B
C
D
< 2% TOC
~21% TOC
~23% TOC
~65% TOC
*
*
*
*
§
§
§
3 2 1
x5
12 11 10 9 8 7 6 5 4 3 2 1 ppm12 11 10 9 8 7 6 5 4 3 2 1 ppm
A
B
C
D
< 2% TOC
~21% TOC
~23% TOC
~65% TOC
A
B
C
D
< 2% TOC
~21% TOC
~23% TOC
~65% TOC
< 2% TOC
~21% TOC
~23% TOC
~65% TOC
*
*
*
*
§
§
§
3 2 1
x5
1= aliphatic species, 2 = predominantly carbohydrates, and 3 = aromatic and amide species. *water or protonated signals because of acids. § denotes DMSO signals. TFA = trifluoroacetic acid
1H-NMR spectra of extracts from the soil (from Buum & Simpson, 2006)
0.1 M NaOD
DMSO-d6
10% TFA in DMSO-d6
5% HSO4 in DMSO-d6
18
Note, DMSO solvent alone and DMSO plus different contents of H2SO4 (v/v) solvent mixtures (2.0 ml) were mixed with dry (10.0 mg of the IHSS Mollisol soil) humin-enriched fine clay, respectively. The mixtures were shaken for 12 h, then left stand overnight. The DMSO extracts (1 ml) diluted with 1 ml distilled water were used for absorbance measurement
DMSO plus acid solvent
Ab
sorb
ance (
40
0 n
m)
Fu
lvic
-lik
e fra
ctio
n
19
Do base/urea and DMSO/H2SO4
change the HS structures?
Our data, based on spectroscopic spectra (FTIR and 13C NMR) for lignin and cellulose, suggests the structural alterations do not occur.
204 0 0 0 3 5 0 0 3 0 0 0 2 5 0 0 2 0 0 0 1 5 0 0 1 0 0 0 5 0 0
a
b
Transm
ittance
Wavelength cm-1
%
Cellulose
Cellulose (DMSO+6%H2SO4 treated)
2898
3351
1638
1430
1373
1320
1170
1109
1063
4 0 0 0 3 5 0 0 3 0 0 0 2 5 0 0 2 0 0 0 1 5 0 0 1 0 0 0 5 0 0
a
b
c
Wavelength cm-1
Transm
ittance
DMSO+6%H2SO4 treated
base/urea treated
original lignin
%
2935
1707
1605
1516
1459
1426
1329
1218
1109
1031
831
21
250 200 150 100 50 0 -5013
C Chemical Shift (ppm)
153
147
134105
114
129
73
55
1430a
b
c
Original lignin
Lignin after base/urea treatment
Lignin after DMSO/H2SO4 treatment
Solid-state 13C NMR of (organosolv) lignin spectra
22
300 250 200 150 100 50 0 -50 -100
300 250 200 150 100 50 0 -50 -100
Maize amended Oak Park soil (12 months) Maize amended Oak Park soil (24 months)
HA pH 12.6
Urea HA
VACP-TOSS 13C NMR CP-TOSS 13C NMR
urea
Full VACP DD
Full VACP DD
23
300 250 200 150 100 50 0 -50 -100
300 250 200 150 100 50 0 -50 -100
300 250 200 150 100 50 0 -50 -100
300 250 200 150 100 50 0 -50 -100
VACP-TOSS 13C NMR spectra and corresponding DD spectra
Urea Fulvic acids
FA pH 12.6
Urea FA
FA pH 12.6
Urea FA
Full VACP DD (thick line)
Full VACP DD (thick line)
Full VACP DD (thick line)
Full VACP DD (thick line)
Clonakilty soil 0-10 cm Clonroche Cultivated soil (0-20 cm)
24
300 250 200 150 100 50 0 -50 -100
300 250 200 150 100 50 0 -50 -100
Full VACP (13 kHz)DD (thick line)
Full VACP (13 kHz)DD (thick line)
100 90 80 70 60 50 40 30 20 10 0 -10
32,30
28,22
25
17,14
100 90 80 70 60 50 40 30 20 10 0 -10
VACP/MAS 13C NMR spectra (13 kHz) and corresponding DD spectra
30,25
Crystalline polymethylene Amorphous polymethylene
CH2 adjacent to ester or acids
25
200 180 160 140 120 100
VACP/MAS 13C NMR spectra (13 kHz) and corresponding DD spectra(expanded spectra) of urea HA, Clonakilty soil 0-10 cm.
155,152,147
135, 129
Lorenz & Preston (2002) suggested 105, 145, 148, 152, 155 for characteristics of lignin and tannin-lignin mixtures.
103
lignin and tannin mixtures?
26
123456789 ppm
Amide/ aromatics
urea
Aliphatic
Aliphatic
Sugars, amino acids, α-oxygen groups
Sugars, amino acids, α-oxygen groups
Amide/ aromatics
DMSO
urea
DMSO
1H NMR spectra of Urea ‘HA’ Clonakilty soil (0-10cm) (1H NMR, lower; the diffusion edited 1H NMR, top).
Diffusione edited
1H NMR
LP
PG
27
Diffusion edited 1H NMR spectra of HALM and DMSO humin from the Mollisol soil.
1 2 3
Urea
45
6
7
DMSO
8*
10
9
11
12
23456789 ppm
LP*Aromatic, amide
Urea
Carbohydrate,
peptides, lignin,
DMSO
Aliphatic
PG
a
b
DMSO humin
HALM** isolated from 0.1 MNaOH + 6 M Urea
† Deuterium exchanged the N-
H to N-D. So the amide resonance in the DMSO humin is strongly attenuated
†
28
• The cell wall in Bacteria contain peptidoglycan, a polymer of N-acetyl glucosamine (NAG), N-acetyl muramicacid (NAM), and amino acids.
• Peptidoglycan: major component in bacterial cell walls.
Identification of N-acetal group (from peptidoglycan) in humic fraction
30
Identification of N-acetal group in peptidoglycan
• A. DMSO FA fraction from DMSO extraction from a grassland soil.
• B. Simulation structure of sugar
and N-acetal part in peptidoglycan,
without the peptide side chains.
9 8 7 6 5 4 3 2 1 ppm
O
H
HH
H
OH
OH
H NH
O
OH
O
H
HH
H
O
H NH
OH
OH
PEP
CO
CH3
C
CH3
O
N
N-H†
CarbohydrateBackbone††
Acetate
A
B
C
9 8 7 6 5 4 3 2 1 ppm
O
H
HH
H
OH
OH
H NH
O
OH
O
H
HH
H
O
H NH
OH
OH
PEP
CO
CH3
C
CH3
O
N
N-H†
CarbohydrateBackbone††
Acetate
A
B
C
DMSO FA
1-D PFGSE-NOESY Selective excitation
31
Identification of R1-OCO-CH2-(CH2)n -R2 methylene unit adjacent to the carbonyl in humic fractions
R1-OCO-CH2-(CH2)n-R2
Lipids
cutin
Waxes
Lipoprotein
Lipopolysaccharide
etc. (e.g. suberin)
32
methylene (CH2)n in aliphatic chains β to an acid or ester
(D) methylene (CH2)n in aliphatic chains (C)
aliphatic chains in cuticular derived material (Simpson et al., 2003a)
33
Urea FA C
1D PFGSE-TOCSY
1D TOE-DE
1H diffusion edited NMR of the
A) a soil urea FA (Clonroche);
B) 1D PFGSE-TOCSY with
selective excitation of peak 1
(simulated spectrum); C) 1D
TOE- DE spectrum with
selective saturated of peak 1.
Assignments:
1, P-OCO-CH2-R methylene
unit adjacent to the carbonyl in
lipids (including lipids,
lipoprotein, lipopolysaccharide
and cutins, etc.); 2, methylene
units in an aliphatic chains, βto an acid or ester; 3,
methylene (CH2)n chain in
aliphatic chains.
selectively saturated CH2
selectively excited CH2
34
2-D 1H-13C HMQC spectrum of DMSO humin
• HMQC: Heteronuclear Multiple Quantum Coherence Spectra.
• Signals are dispersed into 2-D to enhance the assignment of the heavily overlapping proton resonances.
35
2-D 1H-13C HMQC spectrum of DMSO humin
ppm
0.51.01.52.02.53.03.54.04.5 ppm
10
20
30
40
50
60
70
80
6 5
3
4
2
1
DMSO
PG
ppm
ppm
C-H proton in C
CH2 in C
Methoxyl in Lα-proton in P
protons in aliphatics
C: carbohydrate; P: peptide/protein; L: lignin units. LP: lipoprotein; PG: peptidoglycan.
36
Further Consideration
Combination of multi-techniques are desirable in order to get more structural information.
Advanced NMR technique, such as HR-MAS (high resolution magic angel spinning) NMR and TMAH (tetramethylammonium) pyrolysis GC-MSare very suitable for the study of non-soluble SOM samples (e.g. humin, whole soils) and biopolymers.
Using urea enhanced alkaline solvent and DMSO plus small amountof H2SO4 were successfully applied in the study of recalcitrant soil humin material.
We considered that humin would compose of components from plant and microbial origins. Crude humin may compose of humic acids, fulvic acid, and the most recalcitrant humin (mostly consisted of highly aliphatic groups originated from plant and microbial origins).