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EFFECT OF WETTING/DRYING ON THE

CONFORMATIONAL ARRANGEMENT OF A

HETEROGENEOUS ORGANIC MIXTURE AS ASSESSED

BY SOLID STATE 13C NMR SPECTROSCOPY

Università degliStudi di Palermo

P. Conte, A.E. Berns, H. Philipp, P. Burauel, H.-D. Narres, H. Vereecken

Forschungszentrum Jülich

Agrosphere



1. Solid state NMR spectroscopy is a very powerful tool

to analyze insoluble natural organic matter (NOM)2. It is the only way to obtain quantitative information

on the chemical nature of NOM

3. It provides information on the chemical nature of

organic matter directly in bulk soils4. It can be used to study the interactions between

NOM and the inorganic moieties of soils as well asthe interactions with environmental pollutants



Structural unit proposed by Flaig (1960)

Models of humic substances: the macromolecular structures

Structural unit proposed by Stevenson (1982)

Structural unit proposed by Schulten(1993)

Structural unit proposed by Stein(1997)



Models of humic substances: the supramolecular structure

Weak dispersive forces are mainly involved in the conformational stabilization of NOM

Saccharides

Alkyl chains

Aromatic systems

Peptides

Cations

Are the molecular

properties of naturalorganic matter affected bythe experimental handling?

• proposed for the first time by Tschapek, Wasowski and Torres Sanchez, Plant and Soil 63,261-271 (1981)• resumed in Conte and Piccolo, Environmental Science & Technology 33, 1682-1690 (1999)• fully reviewed in Sutton and Sposito, Environmental Science & Technology 39, 9009-9015(2005)



Analyses of the effect of sample preparation on

conformational behavior of some standard organic systems

CH3

CH3

CH3

CH3

CH3

CH3

O H

O

C H3

O HO

CMC

HMB

FERAC

SDS

CH3 O

S

O

O

O Na



Analyses of the effect of sample preparation on

conformational behaviour of some standard organic systems

1. Solid state NMR of pure materials

2. Solid state NMR on dry mixture

3. Solid state NMR on mixture added with water and dried in desiccator (P2O5)

4. Solid state NMR on mixture added with water and freeze dried

Weight ratio 1:1:1:1

Molar ratio 1:52:77:93 for CMC, SDS, FERAC, and HMB, respectively



EXPERIMENTAL SETUP: VCT EXPERIMENTS

-50050100150200250300

∗

−

−−∗

−∗

−∗=

−

CH

CH

CH

T

CT H T

T

H T

CT

H T

T I t I

)(1

exp1)(

exp)(

1)(1

1

1

1

0

ρ

ρ ρ

CT

I(t)

0 1000 2000 3000 4000 5000 6000 7000 8000

4

6

8

1012

14

16

18

20

22

24

26

28

30

32

34

36

38

40

I n t e n s i t y ( a . u . )

Contact time (µµµµs)



EXPERIMENTAL SETUP: VCT EXPERIMENTS

-50050100150200250300

T CH : 1. fast local motions → high T CH values; 2. high amount of protons → low T CH values

T 1 ρ ρρ ρ (H) : proton spin lattice in the rotating frame; fast local motions →

shorterT 1 ρ (H) values. High proton concentration → faster spin diffusion with shorter T 1 ρ (H) values.

Notes : the proton concentration is more important than local molecular mobility in protonated organic systems

VCT experiments overestimate T 1 ρ ( H) values



EXPERIMENTAL SETUP: VSL EXPERIMENTS

Pulse sequence for T 1 ρ ρρ ρ ( (( ( H) measurements

-50050100150200250300

I(t)

t (= delay)

−=

)(

exp)(1

0

H T

t I t I

ρ

0.000 0.005 0.010 0.015 0.020 0.025 0.030 0.035

-200

0

200

400

600

800

1000

1200

1400

I n t e n s i t y

( a . u

. )

delay (s)



50100150

1 7 7

1 0 3 8

3

7 4

6 1

-COOH C1 C4

C2,3,5

C6

Carboxymethylcellulose sodium salt (CMC)



-50050100150200250300

7 4

C2,3,5

Dry mixture

Water suspensionand dried indesiccator

Water suspensionand freeze Dried

Carboxymethylcellulose sodium salt (CMC)

Only the signal of

carbons 2, 3 and 5 isvisible in the spectra ofthe mixtures



CMC

0

20

40

60

80

100

120

pure dry mix dried in desiccator freeze dried

T C H

( u s )

74 ppm

CMC – Results from VCT



CMC

0

20

40

60

80

100

120


T C H

( u s )

74 ppm

CMC – Results from VCT

CMC

2.4

2.5

2.6

2.7

2.8

2.9

3.0

3.1

3.2


T 1 ρ ρρ ρ ( H ) (

m s )

74 ppm

CMC – Results from VSL for T 1ρρρρ(H)



Possible arrangement of CMC in the different conditions

Pure CMC (water amount 11.5%)

CMC

2.4

2.5

2.6

2.7

2.8

2.9

3.0

3.1

3.2

pur e dri ed m ix aft er es sic at or aft er freez e dry ing

T 1 ∠ ∠∠ ∠ ( H ) ( m s )

74ppm

= components = water

Mixing of the dry components(total amount of water 3.3%)

Addition of H2Odesiccator

Hydrogens from residualwater (5.0%) and fromother close molecules

Freeze drying

Hydrogens from residualwater (2.4%) and fromother close molecules



050100150

1 3 1

1 7

CH3

CH3

CH3

CH3

CH3

CH3

Aromatic C

Methyl C

1st order SSB2nd order SSB

Hexamethylbenzene (HMB)



-50050100150200250300

CH3

CH3

CH3

CH3

CH3

CH3

HMB – NOT PRESENT

Aromatic C

Methyl C

Confirmation byHPLC measurements

Hexamethylbenzene (HMB)

Dry mixture

Water suspension

and dried indesiccator




HMB

0

500

1000

1500

2000

2500

3000

131 ppm 17 ppm

T C H

( u s )

puredry

mix

dried

in des.

puredry

mix

dried

in des.

HMB – Results from VCT

HMB

0.0

20.0

40.0

60.0

80.0

100.0

120.0

140.0

160.0

131 ppm 17 ppm

T 1

( H )

( m s )

puredry

mix

dried

in des. pure drymix

driedin des.

HMB – Results from VSL for T 1ρρρρ(H)



Possible arrangement of HMB in the different conditions

π−π interactions

HMB =Addition of other componentsto obtain mixture

HMB

H2O

Addition of water

HMB

desiccatorHMB

Entropy driven compactionof the HMB molecules

Freeze-dried

The π−π interactionsbetween the HMBmolecules are too weak

to prevent removing ofHMB from the mixture



Ferulic Acid

OH

O

CH3

OHO

12

3

4

6

7

89

10 1=126 ppm

2=126 ppm

3=148 ppm

4=149 ppm

5=114 ppm

6=112 ppm

7=56 ppm

8=145 ppm

9=109 ppm

10=173 ppm

50100150

1 7 3

1 4 9

1 4 8

1 4 5

1 2 6

1 1 4

1 1 2

1 0 9

5 6

C10

C4 C3

C8C5

C1, C2 C6C7

C9

Peak assignment done with the aid of dipolar dephasing experiments



Ferulic Acid

-50050100150200250300

OH

O

CH3

OHO

12

3

4

6

7

89

10 1=126 ppm

2=126 ppm3=148 ppm

4=149 ppm

5=114 ppm

6=112 ppm

7=56 ppm

8=145 ppm

9=109 ppm

10=173 ppm

Dry mixture





FERAC

0

100

200

300

400

500

600

700

173 ppm 149 ppm 148 ppm 145 ppm 126 ppm 114 ppm 112 ppm 109 ppm 56 ppm

T

C H

( u s )

pure

dried mix

after essicator

after freeze drying

Ferulic Acid – VCT results (T CH)

FERAC

250.0

300.0

350.0

400.0

450.0

500.0

550.0

600.0

650.0

173 ppm 149 ppm 148 ppm 145 ppm 126 ppm 114 ppm 112 ppm 109 ppm 56 ppm

T 1 r ( H )

pure

dried mix

after essicator

after freeze drying

FERAC – Results from VSL for T 1ρρρρ(H)

dry mix

after desiccator

pure

after freeze drying

dry mix

after desiccator

pure

after freeze drying T 1 ρ

( H ) ( m s )

H R2



Possible arrangement ofFERAC in the differentconditions

OHO

OH

O

CH3

OHO

OH

O

CH3

H R1

H R2

H R3

H R1

H R2

H R3

HO

H

HO

H

HO

H

HO

H

HO

H

HO

H

HO

H

H R1

H R2

H R3

H

O

H

HO

H

OO

OH

O

CH3

HO

H

H

OH

HO

H

HO

H

OO

O

O

CH3

HO

H

H

OH

H

OH

H R1

H R2

H R3

HO

H

OO

OH

O

CH3

HO

H

H

OH

H

OH

addition of components

addition of water

drying in essicator

freeze-drying

slowly relaxing centers

quickly relaxing centers



SDS

102030405060

5 2

3 3

2 5

1 5

C 1C 11 C 12

C 2-10



SDS

-50050100150200250300

Dry mixture



CH3 O

S

O

O

O Na



SDS

0

50

100

150

200

250

300

52 ppm 33 ppm 25 ppm 15 ppm

T C H ( u s ) pure

dried mix

after essicator

after freeze drying

SDS – Results from VCT (T CH)

dry mix

after desiccator

pure

after freeze drying



SDS

0

50

100

150

200

250

300


T C H ( u s ) pure

dried mix

after essicator

after freeze drying

SDS – Results from VCT (T CH)

dry mix

after desiccator

pure

after freeze dryingSDS

0.0

100.0

200.0

300.0

400.0

500.0

600.0


T 1

r ( H ) ( m s ) pure

dried mix

after essicator

after freeze drying

SDS – Results from VSL for T 1ρρρρ(H)

dry mix

after desiccator

pure

after freeze drying T

1 ρ ρρ ρ

( H ) ( m s )



Possible arrangement of SDS in the different conditions

Water amount 1%

OS

O

O

ONa

HH

H

HH

HH

HH

HH

HH

HH

HH

HH

HH

HH

HH Dry mixing

= SDS = other components of the mixture = water

Increase of rigidity

Addition of waterdesiccator

Freeze drying



CONCLUSIONS

Sample handling (wetting and drying) affects molecular properties ofthe organic components of a mixture

Molecular dynamics changes according to the closest proximity of themolecular systems following dissolution and drying at different conditions

Residual water also plays an important role in the relaxationmechanisms

The ππππ−−−−ππππ interactions in the aromatic HMB are not strong enough toprevent removal of such system from the mixture following the freezedrying under forced vacuum conditions

CARE MUST BE USED IN INTERPRETING MOLECULAR DYNAMICSRESULTS FROM NMR EXPERIMENTS ON COMPLEX ORGANICMIXTURES SUCH AS THOSE BELONGING TO EXTRACTED NATURALORGANIC MATTER

THANK YOU FOR YOUR



THANK YOU FOR YOUR

ATTENTION



Purification of

humic substancesby differentcycles of HF/HCltreatments.

Freeze drying

lezione tenuta a juelich (germania) il 05.09.2008

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