The three-dimensional structure of humic substances and soil ...
The three-dimensional structure of humic substances and soil ...
The three-dimensional structure of humic substances and soil ...
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Fresenius J Anal Chem (I995) 351:62-73 Fresenius' Journal <strong>of</strong><br />
© Springer-Verlag 1995<br />
<strong>The</strong> <strong>three</strong>-<strong>dimensional</strong> <strong>structure</strong> <strong>of</strong> <strong>humic</strong> <strong>substances</strong><br />
<strong>and</strong> <strong>soil</strong> organic matter studied by computational analytical chemistry<br />
H.-R. Schulten<br />
Fachhochschule Fresenius, Department <strong>of</strong> Trace Analysis, Dambachtal 20, D-65193, Wiesbaden, Germany<br />
Received: 8 August 1994/Revised: 29 August 1994/Accepted: 31 August 1994<br />
Abstract. A novel <strong>three</strong>-<strong>dimensional</strong> structural concept<br />
for <strong>humic</strong> <strong>substances</strong> <strong>and</strong> <strong>soil</strong> organic matter (SOM) is<br />
proposed which is based on previously published, com-<br />
prehensive investigations combining geochemical, wet-<br />
chemical, biochemical, spectroscopic, agricultural <strong>and</strong><br />
ecological data with analytical pyrolysis. Direct, tempera-<br />
ture-programmed pyrolysis in the ion-source <strong>of</strong> the mass<br />
spectrometer <strong>and</strong> s<strong>of</strong>t ionization in very high electric<br />
fields (Py-FIMS) <strong>and</strong> Curie-point pyrolysis-gas chroma-<br />
tography/mass spectrometry (Py-GC/MS) were the main<br />
applied thermal methods. Emphasis is laid on molecular<br />
modelling <strong>and</strong> geometry optimization <strong>of</strong> complex, poly-<br />
disperse <strong>structure</strong>s <strong>of</strong> biomacromolecules using modern<br />
PC s<strong>of</strong>tware (HyperChem®). Trapping <strong>and</strong> binding <strong>of</strong><br />
atrazine in an organo-mineral complex is introduced as a<br />
first example <strong>of</strong> simulation experiments for <strong>soil</strong> processes<br />
at atomic level (nanochemistry). Future applications <strong>of</strong><br />
semi-empirical calculations <strong>and</strong> molecular dynamics in<br />
pyrolysis studies are outlined.<br />
Introduction<br />
Since practically all pyrolysis data so far have been ob-<br />
tained <strong>and</strong> published as two-<strong>dimensional</strong> (2 D) plots, it is<br />
<strong>of</strong> interest to illustrate recent progress in commercially<br />
available, relatively low cost s<strong>of</strong>tware <strong>and</strong> personal com-<br />
puter (PC) equipment which allow <strong>three</strong>-<strong>dimensional</strong><br />
(3D) displays <strong>and</strong> computer-assisted design (CAD) <strong>of</strong><br />
chemical <strong>structure</strong>s <strong>and</strong> model reactions. In particular<br />
the possibilities for molecular modelling <strong>and</strong> geometry<br />
optimizations <strong>of</strong> complex macromolecules, which are <strong>of</strong>-<br />
ten the target <strong>of</strong> analytical pyrolysis, virtually open up a<br />
new dimension. This is demonstrated in the following for<br />
<strong>humic</strong> <strong>substances</strong>, one <strong>of</strong> the most complex naturally oc-<br />
curring materials, as an example.<br />
Dedicated to Pr<strong>of</strong>essor Dr. Dieter Klockow on the occasion <strong>of</strong> his 60th<br />
birthday<br />
At the 5th International Meeting <strong>of</strong> the International<br />
Humic Substances Society in Nagoya, Japan, a new<br />
approach for a better underst<strong>and</strong>ing <strong>of</strong> the <strong>structure</strong> <strong>of</strong><br />
<strong>humic</strong> <strong>substances</strong> <strong>and</strong> <strong>soil</strong> organic matter (SOM) was in-<br />
troduced [1]. A black <strong>and</strong> white copy <strong>of</strong> the authentic<br />
transparency is shown in Fig. 1, <strong>and</strong> was the basis <strong>of</strong> <strong>three</strong><br />
essential statements.<br />
First, a characteristic portion <strong>of</strong> the organic carbon<br />
consists <strong>of</strong> alkylaromatics <strong>and</strong> forms a flexible carbon-<br />
carbon network <strong>structure</strong>.<br />
Second, the voids formed by this kind <strong>of</strong> <strong>three</strong>-dimen-<br />
sional <strong>structure</strong> could trap <strong>and</strong> bind <strong>three</strong> major classes<br />
<strong>of</strong> organic SOM constituents such as N-containing com-<br />
pounds, aromatics <strong>and</strong> aliphatics.<br />
Third, binding <strong>of</strong> the <strong>humic</strong> <strong>substances</strong> onto the min-<br />
eral surface remains still an unsolved problem (see sche-<br />
matic surface area <strong>and</strong> question mark in the left part <strong>of</strong><br />
Fig. 1).<br />
At that time, typical constituents <strong>of</strong> <strong>humic</strong> <strong>substances</strong><br />
<strong>and</strong> <strong>soil</strong> organic matter in agricultural <strong>soil</strong>s were assem-<br />
~ Agricultural Soil<br />
I . \ / 3<br />
i ~ (CH2)6'"~CH2)>lO OH SOM<br />
CH 3. ~ '~ \ ,CH2-CH<br />
/<br />
] Arom Lic~<br />
@r<br />
Fig. 1. Copy <strong>of</strong> the original co]our transparency shown at the 5th Meet-<br />
ing <strong>of</strong> the International Humic Substances Society, August 6, 1990,<br />
Nagoya, Japan
led for N-containing compounds (Table 1), aromatic<br />
(Table 2) <strong>and</strong> aliphatic compounds (Table 3) which result-<br />
ed from analytical pyrolysis studies using mainly Py-<br />
FIMS <strong>and</strong> Py-GC/MS [1]. More than 170 characteristic,<br />
fundamental <strong>structure</strong>s were identified <strong>and</strong> are listed in<br />
Tables 1 to 3 <strong>and</strong>, considering homologues <strong>and</strong> isomers,<br />
these might well represent > 1000 chemical compounds.<br />
This gave the basis for the proposed carbon-carbon net-<br />
work <strong>structure</strong> <strong>of</strong> <strong>humic</strong> <strong>substances</strong> which has been<br />
visualized with its voids <strong>and</strong> flexible covalent links [2]<br />
<strong>and</strong> clearly demonstrated the complexity <strong>and</strong> variability<br />
<strong>of</strong> the model. <strong>The</strong> latter was particularly emphasized <strong>and</strong><br />
indicated by the % symbols which st<strong>and</strong> for a wide range<br />
Table 1. Nitrogen-containing compounds characteristic for <strong>soil</strong> organic matter in agricultural <strong>and</strong><br />
forest <strong>soil</strong>s (status by Py-FIMS <strong>and</strong> Py-GC/MS, August 6, 1990) [1]. Molecular weights are given<br />
in brackets (m/z)<br />
NH3 ( 17 ) @ CH 3 ~ 0 C--CH3 ~ N CH2-CH20H<br />
HC_=N<br />
1181 (93)<br />
11351 1161)<br />
(27) H3C~-- ~ CHS ~ / C----N<br />
0 H<br />
C~3NH2 ~S/ ~CH ~ 0CH3<br />
(311 (95) 3<br />
~-- CHB ~NH2<br />
~,o %N 2--cH3 113s) OH<br />
H--C~-NH 2 11631<br />
H3C C~.NH 2 H 1951 C~2--CH20H 11711<br />
1591<br />
~ OH 1137) ~CO--CH 3<br />
~ C2Hs ~N'-CH3<br />
H (95) H<br />
(67) H (1731<br />
H N ~ CH3<br />
(68) 2 H CH 3 (175)<br />
u 0 ~C00H<br />
(79)<br />
(113)<br />
--CN<br />
OH<br />
0%C/H<br />
(149) ~/~N'~CH3<br />
H<br />
( 175 )<br />
CH3 ~ CH3 CH2--0<br />
(811 (1171<br />
H N~ 3Hv<br />
+=0<br />
CH3 ~ CH3<br />
• 181) (124) (185)<br />
~OH ~ N N'\0CH3 C12H25-C-N<br />
1152) (195)<br />
H<br />
(55) . k-.
64<br />
Table 2. Aromatic compounds characteristic for <strong>soil</strong> organic matter (see Table 1) [1]<br />
CH=CH 2<br />
H3C0 OCH 3<br />
(78) v\CH3 0H<br />
1118) 1134)<br />
(154)<br />
O%C--H ~J_C~N CH3<br />
H 3 OH<br />
(94) ~ CN2-CH3 (CH2)4-CH3<br />
(104)<br />
CH3 Y "0CH3<br />
H3 OH CH=CH-CHa<br />
OCH 3<br />
1120) 0CH 3<br />
(106) CH 3<br />
CH3_C=CH 2<br />
H 3 CH2-CH3 (CH3)2 OCH 3<br />
1106) OH 1164)<br />
(144) OC--CH 3<br />
0CH3 1146) OCH3<br />
(106) OH OH<br />
~OH ~CH3 (124) ~--~CH2)3-CH 3 ~(164)<br />
1108) (130) 1148) 1166)<br />
CH3 CH 3 - CH=CH2 ~3<br />
OH OH<br />
(116) y "OCH 3<br />
(134) OH<br />
ed as a first draft 2D <strong>structure</strong> [3]. It had the elemental<br />
composition <strong>of</strong> C308H328090N 5 with a molecular mass <strong>of</strong><br />
5 540.027 g tool -1 <strong>and</strong> an elemental analysis <strong>of</strong> 66.8% C,<br />
6.0% H, 26.0°70 O, 1.3% N. Compared to isolated stan-<br />
dard HAs with about 56.3070 C <strong>and</strong> 32.8% O, this pro-<br />
posed HA <strong>structure</strong> is obviously too high in carbon <strong>and</strong><br />
too low in oxygen. However, carbohydrates have been re-<br />
ported to account for about 10% <strong>of</strong> the HA weight <strong>and</strong><br />
a similar value has been suggested for proteinaceous ma-<br />
terials in HA [3]. Thus, it has been assumed that one HA<br />
molecule traps within its voids approximately 10% carbo-<br />
hydrates <strong>and</strong> 10% proteinaceous materials. <strong>The</strong> resulting<br />
HA complex has then an elemental composition <strong>of</strong><br />
C342H3880124N12 with a molecular mass <strong>of</strong> 6650.912g<br />
mo1-1 <strong>and</strong> an elemental analysis <strong>of</strong> 61.8% C, 5.9% H,<br />
_CH3 (CHe)s-CH3<br />
(152) 1176)<br />
29.8% O, 2.5% N. If more carbohydrates <strong>and</strong>/or pro-<br />
teinaceous materials are added, the C content decreases<br />
<strong>and</strong> the O content increases.<br />
Thus, for a stereochemical underst<strong>and</strong>ing <strong>of</strong> the<br />
<strong>structure</strong> <strong>of</strong> <strong>humic</strong> <strong>substances</strong> <strong>and</strong> their reactions with<br />
biological <strong>and</strong> anthropogenic compounds <strong>three</strong> main,<br />
urgent questions emerged:<br />
1. What is a fast, reliable <strong>and</strong> versatile method to convert<br />
the 2D <strong>structure</strong> into a 3D <strong>structure</strong>?<br />
2. Is it possible to optimize the geometry <strong>of</strong> the chemical<br />
3 D <strong>structure</strong> <strong>and</strong> to determine <strong>and</strong> minimize its en-<br />
ergy?<br />
3. How can chemical reactions <strong>of</strong> such complex biomac-
Table 2 (continued)<br />
o<br />
CH2-CH2-C~H<br />
0H<br />
CH2-CH:-CHO<br />
H3C0 OCH 3<br />
OCH3 OH<br />
(210)<br />
CH=CH2 (180) 22--(CH2) s-CH3<br />
H3CO~0CH 3 ~ (218)<br />
OKcI{2__CH3(180) ~ ~CH2) 7-cn3<br />
/ ~ 0H (220)<br />
H3CO OCH 3<br />
OH ~H2--( CH2 ) 9-CH3<br />
1182)<br />
©<br />
(232)<br />
CH2--( CH 2 ) io-CH3<br />
OH OK (186) ~--~H3<br />
--( CH 2 ) ~-CH3<br />
(246 )<br />
(1901 ~ -(CH2) II--CH3<br />
H O ~ 12601<br />
1194) ~C6H14<br />
CH2-CHO<br />
TC6HI4<br />
H3C0 0CH 3 (264)<br />
OH<br />
(196) ~--(CH2) 12-CH3<br />
.~--( CH2 ) 7 -CH3<br />
(204)<br />
(274)<br />
¢<br />
CH2--(CH2) 13-CH3<br />
12881<br />
-4CH 2114-CH3<br />
(302)<br />
-qCH 2 ) 15-CH3<br />
1316)<br />
-~CH 2 ) 16-CH3<br />
(330)<br />
romolecules be simulated at atomic <strong>and</strong> molecular<br />
level?<br />
Experimental<br />
Pyrolysis-fieM ionization mass spectrometry (Py-FIMS)<br />
For temperature-resolved Py-FIMS, about 100~tg <strong>of</strong><br />
<strong>humic</strong> <strong>substances</strong> such as <strong>humic</strong> acid (HA), fulvic acid<br />
(FA), humin or 5 mg <strong>of</strong> whole <strong>soil</strong> samples, respectively,<br />
were thermally degraded in the ion source <strong>of</strong> a MAT 731<br />
(Finnigan, 28127 Bremen, Germany) modified high-per-<br />
formance (AMD Intectra GmbH, 27243 Harpstedt, Ger-<br />
many) mass spectrometer. <strong>The</strong> samples were weighed be-<br />
Table 3. Aliphatic compounds characteristic for <strong>soil</strong> organic matter<br />
(see Table i) [11<br />
CH2=CHCHO<br />
CH3-C0--CH 3<br />
CH3-C00H<br />
CHa--CO--CHO<br />
CH2-C--CO--CH 3<br />
CN3-CH--CH--CH--CH--CH 3<br />
3 H C ~ CH3<br />
65<br />
(56)<br />
(58)<br />
(601<br />
(72)<br />
(84)<br />
(96)<br />
0<br />
(i12)<br />
Qo 1114)<br />
3HC-~ OH<br />
11281<br />
0 0 0<br />
II II II<br />
CH3-C--CH2-C--CH2-C--CH 3<br />
11421<br />
~H3 ?H3 ~H3<br />
CH3-CH--(CH2) 3--CH--(CH2) 3-CH--CH2=CH--CH2 (224)<br />
CH CH 3 CH 3<br />
3 i i<br />
CH3--CH--~CH2)3--CH---~CH 2 3~CH~--~CH2 2-C~3<br />
CH3--~ CH 2 )IT"CH20H<br />
CH 3 CH 3 CH 3 CH 3<br />
I I I<br />
CH3-CK--~CH 2 3-CN--CH 2 3-CH--CN 2 3-C=CH2<br />
n=2-30 series<br />
CH3-~ CH 2 )~--CH3<br />
n=i-28 series<br />
CH2=CH--( CH 2 )~--CH 3<br />
CH3--( CH 2 )~4 COOH<br />
(226)<br />
12281<br />
(266)<br />
(58-450)<br />
(56-4341<br />
(256)<br />
14101<br />
fore <strong>and</strong> after Py-FIMS (error + 0.01 mg) to determine the<br />
pyrolysis residue <strong>and</strong> the produced volatile matter. <strong>The</strong><br />
heatable/coolable direct introduction system with elec-<br />
tronic temperature-programming, adjusted at the + 8 kV<br />
potential <strong>of</strong> the ion source <strong>and</strong> the field ionization emit-<br />
ter, was used. <strong>The</strong> slotted cathode plate serving as count-<br />
er electrode was on - 6 kV potential. Thus, at 2 mm dis-<br />
tance between the emitter tips <strong>and</strong> the cathode, in total a<br />
potential difference <strong>of</strong> 14 kV is applied resulting in an ex-<br />
tremely high electric field strength which is the essential<br />
basis for s<strong>of</strong>t ionization. All samples were heated in high<br />
vacuum (1.3 × 10 -4 Pa) from 323 K to 973 K at a heating<br />
rate <strong>of</strong> approximately 0.5Ks -1. About 60magnetic
66<br />
(CH3)o-3<br />
(CHs)o-2<br />
0 ~<br />
@O ~<br />
(CH3) 0-5 @<br />
OH<br />
H O~'.1<br />
oH°'f -° oH°-.-r "°H ,o,<br />
OH OH<br />
scans were recorded for the mass range 16 to 1000<br />
Daltons. In general, at least <strong>three</strong> replicates were per-<br />
formed for each sample. <strong>The</strong> total ion intensities (TII) <strong>of</strong><br />
the single spectra were normalized to 1 mg sample weight,<br />
averaged for replicate runs, <strong>and</strong> plotted versus the<br />
pyrolysis temperature, resulting in Py-FIMS thermo-<br />
grams. For the selection <strong>of</strong> biomarkers <strong>and</strong> quantitative<br />
evaluations, in particular <strong>of</strong> whole <strong>soil</strong>s <strong>and</strong> <strong>soil</strong> particle-<br />
size fractions, detailed descriptions <strong>of</strong> the method have<br />
been given recently [6-9].<br />
HO<br />
OH<br />
Curie-point pyrolysis-gas chromatography~mass<br />
spectrometry (Py-GC/MS)<br />
<strong>The</strong> <strong>humic</strong> <strong>substances</strong> <strong>and</strong> <strong>soil</strong>s were pyrolyzed in a type<br />
0316 Curie-point pyrolyzer (Fischer, 53340 Meckenheim,<br />
Germany). <strong>The</strong> samples were not pretreated except drying<br />
<strong>and</strong> milling. <strong>The</strong> final pyrolysis temperatures (Tf) em-<br />
ployed were 573 K, 773 K <strong>and</strong> 973 K, respectively. <strong>The</strong> to-<br />
tal heating time (THT) was varied between 3 <strong>and</strong> 9.9 s.<br />
Following split injection (split ratio 1:3; flow rate<br />
1 ml 20 s -1) the pyrolysis products were separated on a<br />
gas chromatograph (Varian 3700, 64289 Darmstadt, Ger-<br />
many), equipped with a 30 m capillary column (DB5),<br />
coated with 0.25 gm film thickness <strong>and</strong> an inner diameter<br />
<strong>of</strong> 0.32 mm. <strong>The</strong> starting temperature for the gas chro-<br />
matographic temperature program was 313 K, <strong>and</strong> the<br />
end temperature was 523 K, with a heating rate <strong>of</strong><br />
10 K min -1. <strong>The</strong> gas chromatograph was connected to a<br />
nitrogen-selective, thermosensitive detector (TSD) <strong>and</strong> a<br />
double-focusing Finnigan MAT 212 mass spectrometer.<br />
Conditions for mass spectrometric detection in the elec-<br />
tron ionization mode were +3 kV accelerating voltage,<br />
70eV electron energy, 2.2kV multiplier voltage, 1.1 s/<br />
OH<br />
HO<br />
HO<br />
(CH3)0-2<br />
/.(N ~.,.,2 (CH3)0-2<br />
H<br />
C=N<br />
OH<br />
~0/ ~C_. ?<br />
O<br />
HO =N<br />
[~OH /CH2OH Fig. 2. Drawing <strong>of</strong> the proposed<br />
<strong>humic</strong> acid <strong>structure</strong> using Indian<br />
ink <strong>and</strong> a chemical stencil as published<br />
in Ref. [3]<br />
mass decade scan speed <strong>and</strong> a recorded mass range be-<br />
tween m/z 50 <strong>and</strong> m/z 500. A detailed description <strong>of</strong> the<br />
principle, potential <strong>and</strong> limitations <strong>of</strong> Py-GC/MS <strong>of</strong><br />
<strong>humic</strong> fractions <strong>and</strong> <strong>soil</strong>s has been given [10]. Further-<br />
more, studies <strong>of</strong> organic nitrogen-containing compounds<br />
in <strong>soil</strong>s using the TSD detector were reported [11] which<br />
exp<strong>and</strong>ed the knowledge on the <strong>structure</strong> <strong>of</strong> 'unknown'<br />
<strong>soil</strong> nitrogen beyond the status reported in Tables 1- 3.<br />
Structural modelling <strong>and</strong> geometry optimization<br />
For all described 2D- <strong>and</strong> 3 D-work, model construction,<br />
chemical interaction studies <strong>and</strong> semi-empirical calcula-<br />
tions, the HyperChem ® s<strong>of</strong>tware (release 2 for windows)<br />
[12] was used which is assisted by a comprehensive manu-<br />
al. In the present text some main s<strong>of</strong>tware comm<strong>and</strong>s are<br />
indicated in brackets (in italics). <strong>The</strong> program is supplied<br />
by Computer Aided Animation (CAA) GmbH, Kenner-<br />
weg 15, 72622 Ntirtingen, Germany.<br />
<strong>The</strong> employed PC consisted <strong>of</strong> the Escom local-bus<br />
tower 486DX2/66, VLB 34 in combination with 8 MB<br />
memory, Escom 17" colour monitor with AVGA VL<br />
bus/1MB graphic card, Samsung 250 MB disk, 250 MB<br />
powerstreamer, <strong>and</strong> peripheric hardware plus utility pro-<br />
grams (Escom <strong>of</strong>fice, Rheinstr. 41, D-65185 Wiesbaden,<br />
Germany).<br />
Results <strong>and</strong> discussion<br />
Building molecules<br />
Building a 3 D model <strong>of</strong> the 2D <strong>humic</strong> acid <strong>structure</strong><br />
(Fig. 2) is performed in five steps using the HyperChem ®<br />
s<strong>of</strong>tware.
First, a preliminary 2 D <strong>structure</strong> <strong>of</strong> the C-C skeleton<br />
is drawn by h<strong>and</strong> into the workspace using the drawing<br />
tool <strong>and</strong> is far from perfect as shown in Fig. 3. In addi-<br />
tion to carbon, the elements oxygen <strong>and</strong> nitrogen are in-<br />
cluded (Default Element). In this manner 308 carbon-,<br />
90 oxygen- <strong>and</strong> 5 nitrogen-atoms (403 atoms in total) are<br />
positioned.<br />
Second, hydrogen is attached with accurate bond<br />
lengths <strong>and</strong> angles to the preliminary <strong>structure</strong> using the<br />
s<strong>of</strong>tware (Add Hydrogen) as shown in Fig. 4. Whereas in<br />
the original publication the 2 D HA subunit <strong>structure</strong> [2]<br />
was incomplete <strong>and</strong> was supposed to have a wide variety<br />
<strong>of</strong> different bonding sequences (see :k-- symbols), the mol-<br />
ecule in Fig. 4 represents a defined, intact HA monomer<br />
(738 atoms). Thus, in order to obtain a complete chemi-<br />
cal compound, it had to be supplemented by 7 hydrogen<br />
atoms. <strong>The</strong> corresponding elemental composition then is<br />
C308H335090N 5 with a molecular mass <strong>of</strong> 5547.004g<br />
mol -~ <strong>and</strong> an elemental analysis <strong>of</strong> 66.69% C, 6.09% H,<br />
25.96% O, <strong>and</strong> 1.26% N.<br />
Third, building the 3 D <strong>structure</strong> <strong>of</strong> the selected HA<br />
molecule (Model BuiM) with accurate bond distances<br />
<strong>and</strong> bond angles, torsion angles, van der Waals forces,<br />
<strong>and</strong> hydrogen bonds, is illustrated in Fig. 5. This prelimi-<br />
nary, energetically still unoptimized <strong>structure</strong> shows<br />
clearly the atomic distances <strong>and</strong> angles (Sticks) <strong>and</strong> gives<br />
a first <strong>three</strong>-<strong>dimensional</strong> <strong>structure</strong> information. Even this<br />
rough display indicates already the importance <strong>of</strong><br />
aliphatic chains as link between the aromatic moieties<br />
<strong>and</strong> prerequisite for the formation <strong>of</strong> gaps <strong>and</strong> voids in<br />
the HA <strong>structure</strong>.<br />
Fourth, the (Sticks) <strong>structure</strong> in Fig. 5 is converted in-<br />
to the 3 D <strong>structure</strong> (Disks, Perspective) shown in Fig. 6<br />
which better reflects the space requirements <strong>of</strong> the 738 at-<br />
oms. <strong>The</strong> white disks are hydrogen atoms, black disks ni-<br />
trogen, the brighter shaded disks are carbons <strong>and</strong> the<br />
darker ones oxygen. It is evident that even more complex<br />
<strong>structure</strong>s such as naturally occurring organomineral<br />
complexes or trapping <strong>of</strong> biological <strong>and</strong> anthropogenic<br />
<strong>substances</strong> in SOM will be difficult to evaluate in these<br />
Fig. 3. H<strong>and</strong>drawn two-<strong>dimensional</strong> version (2D) <strong>of</strong> the <strong>humic</strong> acid<br />
draft <strong>structure</strong> using the drawing tool in the HyperChem ® workspace<br />
-2<br />
Fig. 4. Hydrogen attachment to the h<strong>and</strong>drawn <strong>humic</strong> acid draft struc-<br />
ture performed by HyperChem ® (Add Hydrogen)<br />
black <strong>and</strong> white plots. As will be illustrated in the follow-<br />
ing fifth step, colourplots can be <strong>of</strong> great help <strong>and</strong> allow<br />
a distinct view <strong>and</strong> facilitated evaluation <strong>of</strong> the complex,<br />
large macromolecules.<br />
Chemical calculations<br />
After building the molecule from scratch (Fig. 3), the<br />
atomic coordinates in the s<strong>of</strong>tware files allow a wide<br />
range <strong>of</strong> quantitative evaluations. Measuring structural<br />
properties includes the determination <strong>of</strong> bond distances,<br />
bond angles, torsion angles, non-bonded distances, <strong>and</strong><br />
hydrogen bonds. For minimizing the total energy <strong>of</strong> the<br />
molecular system <strong>and</strong> thus increasing the stability <strong>of</strong> its<br />
conformation, step-by-step instructions for performing<br />
single point calculations, geometry optimization, <strong>and</strong><br />
molecular dynamics are given by HyperChem ®.<br />
When one cycle <strong>of</strong> a single-point calculation <strong>of</strong> the<br />
HA 3 D <strong>structure</strong> in Fig. 6 was performed using the algo-<br />
rithm <strong>of</strong> Polak-Ribiere (conjugate gradient), the total en-<br />
Fig. 5. Three-<strong>dimensional</strong> <strong>structure</strong> (3D) <strong>of</strong> the draft HA <strong>structure</strong><br />
shown in Fig. 4 using HyperChem ® for molecular modelling (Build<br />
Model). Display <strong>of</strong> the <strong>structure</strong> in the sticks mode (Rendering; Sticks)<br />
67
68<br />
ergy <strong>of</strong> the molecular system was calculated with<br />
6993.92 kJ 0.1 nm -1 mo1-1 <strong>and</strong> a gradient (derivative <strong>of</strong><br />
the energy with respect to all Cartesian coordinates) <strong>of</strong><br />
91.16 kJ 0.1 nm -1 mol -I. <strong>The</strong> termination conditions<br />
for this calculation were 83.80 kJ nm- 1 mol - 1 <strong>and</strong> 11070<br />
cycles maximum. After approximately 4700 calculation<br />
Fig. 6. First approximation (1 cycle, 3 points, convergence limit <strong>of</strong><br />
83.80 kJ 0.1 nm -1 mol ~) for the geometrical optimization <strong>of</strong> the HA<br />
<strong>structure</strong> (II) given in Fig. 5 (Compute; Single Point; Geometry Optimi-<br />
zation) by semi-empirical calculations. Display <strong>of</strong> the <strong>structure</strong> in the<br />
disks mode (Rendering; Disks)<br />
~ J<br />
Fig. 7. Geometry optimization<br />
<strong>and</strong> energy minimization <strong>of</strong> the<br />
3 D <strong>structure</strong> <strong>of</strong> HA (II, Fig. 6)<br />
after 4700 calculation cycles <strong>and</strong><br />
a resulting total energy <strong>of</strong><br />
710.70 kJ nm -1 mo1-1 <strong>and</strong> a<br />
convergence gradient <strong>of</strong><br />
0.037 kJ nm- 1 mol- 1<br />
cycles with a convergence limit <strong>of</strong> 0.83 kJ 0.1 nm -1<br />
tool-l, the energy minimization leads to a molecular en-<br />
ergy <strong>of</strong> 752.57kJ 0.1 nm -1 tool -1 with a gradient <strong>of</strong><br />
0.71 kJ 0.1 nm -1 tool -1. As an illustration <strong>of</strong> the energy<br />
minimization process in Fig. 7 the corresponding HA<br />
<strong>structure</strong> is displayed. Rotation <strong>of</strong> the energy-minimized
HA 3 D version demonstrates the flexible network with<br />
voids <strong>and</strong> hollows which <strong>of</strong>fer binding <strong>and</strong> trapping <strong>of</strong><br />
biological <strong>and</strong> anthropogenic molecules [2]. Further-<br />
more, long-continued calculation periods (> 6 days) give<br />
energies in the order <strong>of</strong> 608 kJ 0.1 nm-1 mol-1 <strong>and</strong> gra-<br />
dients
70<br />
Fig. 9. Result <strong>of</strong> atrazine transport into the 3 D structural model <strong>of</strong> an organo-mineral complex in <strong>soil</strong> with atrazine trapped in the voids (V)<br />
the atrazine molecule moves with decreasing energy <strong>and</strong><br />
increasing conformational stability step-by-step more<br />
into the available space <strong>of</strong> the void. Finally, arriving with<br />
the geometry optimization <strong>and</strong> energy minimization<br />
close to a total energy <strong>of</strong> 10008.21 kJ nm -1 mo1-1 <strong>and</strong> a<br />
gradient <strong>of</strong> 0.37 kJ nm-1 mol-t, the atrazine molecule is<br />
immobilized in the void by formation <strong>of</strong> a hydrogen bond<br />
from H (21, molecule I) to a HA carboxyl group <strong>of</strong> II (O<br />
(606, molecule II)) which is an integral part <strong>of</strong> the<br />
organo-mineral particle V now. Figure 10c shows the<br />
space requirements <strong>of</strong> the atrazine molecule in the hollow<br />
space, but also, may be more important, how much space<br />
is left for chemical decomposition reactions.<br />
Nanochemistry<br />
For the biological <strong>and</strong> chemical reactivity <strong>of</strong> the trapped<br />
anthropogenic compound it is highly interesting to deter-<br />
mine exactly the distances between I <strong>and</strong> the surrounding<br />
molecular complex <strong>of</strong> the HA II <strong>and</strong> the silica matrix IH.<br />
As derived from Fig. 10a, the numbered atoms <strong>and</strong><br />
Fig. 10b the atoms labelled by element symbols, some
~749 ~54<br />
Fig. 10a-e. <strong>The</strong> enlarged section <strong>of</strong> a void in an organo-mineral com-<br />
plex (V; geometry <strong>and</strong> energy optimization: bonds <strong>and</strong> angles, torsions,<br />
van de Waals forces, hydrogen bridges) is shown in which atrazine is<br />
trapped <strong>and</strong> fixed via a distinct hydrogen bond <strong>and</strong> SOM is bound to<br />
the mineral matrix via hydrogen bonding in addition to <strong>three</strong> covalent<br />
bonds, a Section (Sticks, Atoms, Number) is shown with the displayed<br />
selected distances between atrazine I <strong>and</strong> II <strong>and</strong> III are as<br />
follows:<br />
Starting from C1 (i, I) (atom 1, molecule I) the dis-<br />
tances to H (256, III) are 0.7947 nm (HyperChem ® out-<br />
put 7.947477 Angstrom), to H (255, III) 1.0466 nm, to O<br />
(225, III) 0.3737 nm, O (187, III) 0.3431 nm, O (100, III)<br />
0.8717 nm <strong>and</strong> to the hydrogens <strong>of</strong> the long aliphatic<br />
chain (see lower part <strong>of</strong> V in Fig. 9) H (1004, II)<br />
0.6051 nm <strong>and</strong> H (1011, II) 0.8632 nm. From H (3, I) to<br />
H (950, II) a distance <strong>of</strong> 0.3074 nm, to H (996, II) <strong>of</strong><br />
0.2160 nm <strong>and</strong> to H (1004, II) <strong>of</strong> 0.4177 nm was measured<br />
for the lower part <strong>of</strong> the void. In the upper part, for in-<br />
stance between H (17, I) <strong>and</strong> H (902, II) 0.3074 nm, H<br />
(749, II) 0.4468 nm, H (745, II) 0.6537 nm, C (400, II)<br />
0.7601 rim, O (577, II) 0.4885 nm were obtained. Despite<br />
the apparent proximity between H (19, I) <strong>and</strong> H (251, III)<br />
a distance <strong>of</strong> 1.1614 nm was found <strong>and</strong> clearly demon-<br />
strated the necessity <strong>of</strong> exactly calculated coordinate<br />
systems. Moreover, the distance between H (21, I) <strong>and</strong><br />
O (606, II) was <strong>of</strong> interest, because it narrowed with<br />
progressing geometry optimization from >1rim to<br />
0.2221 nm generating a hydrogen bond which immobi-<br />
lized the atrazine molecule in the HA void. <strong>The</strong> more the<br />
total energy content <strong>of</strong> the organo-mineral complex V is<br />
/<br />
1<br />
6<br />
4<br />
6~4 "~<br />
71<br />
atoms labelled by numbers; b Section (Sticks, Atoms, Symbols) is<br />
shown with atoms labelled by element symbols; c Section (Rendering;<br />
Disks, Perspective) is shown as colour plot illustrating the space re-<br />
quirements <strong>of</strong> the pesticide <strong>and</strong> the available <strong>three</strong>-<strong>dimensional</strong> trap-<br />
ping space<br />
lowered, the more shrinking <strong>of</strong> the hollow space <strong>and</strong> con-<br />
sequently stronger bonding <strong>of</strong> the atrazine is observed. In<br />
summary, one can assume that without operator interfer-<br />
ence energy minimization simulates aging processes.<br />
<strong>The</strong> results <strong>of</strong> the <strong>three</strong>-<strong>dimensional</strong> <strong>structure</strong> <strong>and</strong> dis-<br />
tance measurements furthermore allow to evaluate the<br />
space between the trapped molecule <strong>and</strong> its host, the<br />
organo-mineral complex. In particular the space filling <strong>of</strong><br />
trapped biological or anthropogenic <strong>substances</strong> is sup-<br />
posed to have an essential influence on their presumable<br />
fate in SOM. Clearly air (N2, O2) <strong>and</strong> water (HzO) <strong>and</strong><br />
small organic acids (CH3COOH) can invade the gaps be-<br />
tween the pesticide <strong>and</strong> organic or inorganic <strong>soil</strong> surfaces.<br />
Thus, non-metabolic decomposition processes are easily<br />
possible at this stage. Metabolic processes, however, can<br />
be excluded as microorganisms such as bacteria (> 200 to<br />
1000 nm) <strong>and</strong> fungi (> 10000 nm) have no access into the<br />
described voids <strong>and</strong> even enzymes are too large to react<br />
with the trapped materials when the front <strong>and</strong> back side<br />
<strong>of</strong> the void is closed by recalcitrant SOM or inorganic<br />
<strong>substances</strong>. <strong>The</strong> latter is much more likely, as the organic<br />
matter in agricultural <strong>soil</strong>s is only around 3°70. Thus, in<br />
nature the inorganic matrix represented here by the<br />
hypothetic model III should be larger by at least a factor<br />
<strong>of</strong> 30.
72<br />
Fig. 10b<br />
\<br />
Fig. 10c
It is expected that computational chemistry in combi-<br />
nation with analytical pyrolysis (<strong>and</strong> analytical chemistry<br />
in general) will play an important role in future studies<br />
not only in environmental studies [14] <strong>and</strong> ecology [15]<br />
but also in <strong>soil</strong> science [16] <strong>and</strong> agriculture as described<br />
recently [13, 17].<br />
Conclusions<br />
(1) <strong>The</strong> molecular-chemical <strong>structure</strong> <strong>of</strong> <strong>soil</strong> organic mat-<br />
ter is mainly determined by the inorganic <strong>soil</strong> matrix, the<br />
climate, plant precursors, <strong>and</strong> the dynamics <strong>and</strong> specific<br />
activity <strong>of</strong> biomass.<br />
(2) <strong>The</strong> resulting <strong>three</strong>-<strong>dimensional</strong>, organo-mineral<br />
complexes <strong>of</strong> decomposition products, biologically syn-<br />
thesized materials <strong>and</strong> <strong>soil</strong> mineral particles can be ana-<br />
lyzed indirectly by Py-GC/MS <strong>and</strong> directly by in-source<br />
Py-MS.<br />
(3) Analytical pyrolysis <strong>of</strong> <strong>humic</strong> <strong>substances</strong> <strong>and</strong><br />
whole <strong>soil</strong>s yields a vast variety <strong>of</strong> pyrolysis products.<br />
<strong>The</strong>ir intra- <strong>and</strong> intermolecular chemical properties are<br />
only understood when fast, reliable <strong>and</strong> powerful com-<br />
puter-assisted data h<strong>and</strong>ling <strong>and</strong> evaluating allows the<br />
chemistry to evolve in the <strong>three</strong> dimensions <strong>of</strong> space un-<br />
der the precise structural parameters such bond lengths,<br />
angles, <strong>and</strong> torsions, van der Waals forces, hydrogen<br />
bridges, etc.<br />
(4) To reassemble the identified chemical subunits to<br />
a hypothetic <strong>structure</strong> <strong>of</strong> <strong>soil</strong> organic matter that reflects<br />
as closely as possible the genuine status, above all con-<br />
structive interdisciplinary work <strong>and</strong> collaboration is<br />
needed.<br />
Acknowledgements. <strong>The</strong> author is grateful for the financial support by<br />
the Deutsche Forschungsgemeinschaft (projects Schu 416/3; Schu 416/<br />
18-1) <strong>and</strong> the Ministry <strong>of</strong> Science <strong>and</strong> Technology, Bonn-Bad Godes-<br />
berg, <strong>and</strong> the Umweltbundesamt, Berlin, Germany. Thanks are due to<br />
R. Hempfling, R. Mt~ller, C. Sorge <strong>and</strong> H. Wilken (Fresenius Group)<br />
<strong>and</strong> P. Leinweber, University <strong>of</strong> Osnabrack for their help <strong>and</strong> coopera-<br />
73<br />
tion. To work with the "pioneer <strong>of</strong> <strong>humic</strong> <strong>substances</strong>" Dr. Morris<br />
Schnitzer, Agriculture Canada, Ottawa, has been <strong>and</strong> is a unique occa-<br />
sion which gives its merits <strong>and</strong> rewards by itself.<br />
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