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Urea Enhanced NaOH Solvent and H2SO4

Enhanced DMSO Solvent System in The Study of Humic Substances

Department of Chem. & Environ. Sciences

[Guixue Song]

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Traditional alkaline extraction procedure for isolation of humin

HF/HCl de-ash

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

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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.

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• 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

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•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.

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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

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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

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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)

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• 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).

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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

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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)

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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

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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)

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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

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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

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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

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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

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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

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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)

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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

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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

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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?

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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

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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

†

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• 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

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The peptidoglycan cross linking with other peptidoglycan chains form a complex layer

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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

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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)

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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)

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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

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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.

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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.

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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).

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Thanks for your attention! Questions?