Natural Resources, 2012, 3, 29-34
http://dx.doi.org/10.4236/nr.2012.32005 Published Online June 2012 (http://www.SciRP.org/journal/nr)
29
Effect of the Pre-Treatment Severity on the Antioxidant
Properties of Ethanol Organosolv Miscanthus x giganteus
Lignin
Roland El Hage1,2, Dominique Perrin3, Nicolas Brosse3*
1Centre des Matériaux de Grande Diffusion (CMGD), Ecole des Mines d’Alès, France; 2INNOBAT, Cap Alpha, Clapiers, France;
3Laboratoire d’Etude et de Recherche sur le MAteriau Bois, Faculté des Sciences et Techniques, Nancy-Université, Vandœuvre-
lès-Nancy, France.
Received November 7th, 2011; revised December 29th, 2011; accepted January 8th, 2012
ABSTRACT
The effect of the severity of an organosolv treatment of Miscanthus x giganteus on antioxidant capacity of the obtained
lignin was studied. Four organosolv lignins extracted with different severity conditions were chosen and tested. Results
obtained using the methyl linoleate method have shown a correlation between oxygen uptake index and the combined
severity. It was found that lignin extracted at higher severity pre-treatment and with a higher phenolic hydroxyl content,
lower aliphatic hydroxyl content, molecular weight and polydispersity has the highest antioxidant capacity.
Keywords: Miscanthus x giganteus; Organosolv Lignin; Antioxidant; Combined Severity; Molecular Weight;
Phenolic and Aliphatic Hydroxyl
1. Introduction
The organosolv pulping has been the subject of con-
siderable research activity and has generated increasing
interest as the pulp and paper industry is moving toward
minimization of environmental impact [1]. In the 1990’s,
this technology was developed at the industrial scale in
Canada (Alcell® Process). One of the advantages of the
organosolv process is the fractionation of the lignocel-
lulosic materials into three major components: cellulosic
fibers, hemicelluloses and lignin. As a result, with the
increasing attention devoted to the biorefinery concept, a
renewed interest in the organosolv treatment is currently
observed and this technology seems to be promising for
the production of ethanol and high value chemicals and
materials from lignin, hemicelluloses and extractives [2].
Indeed, production of high-quality lignin is one of the
unique advantages of the organosolv treatment over alter-
native processes which generally produce a degraded
lignin employed in low added value applications and
energy production. Oganosolv lignins are high-purity,
low molecular weight and sulfur-free products. Moreover,
they are soluble in many organic solvents, possess low
glass transition temperatures, and are easier to thermally
process than kraft lignins. Thus, availability of such high-
quality lignin in large quantities should stimulate deve-
lopment in new lignin applications in the fields of fibers,
biodegradable polymers, adhesives... Lignins as well as
other polyphenols are potent free radical scavengers [3]
and organosolv lignins are considered to be a valuable
source of antioxidant phenolic compounds, which could
be recovered as functional food or feed ingredients. A
large number of biochemical or chemical methods are
used to measure antioxidant capacity. One class of those
methods is based upon inhibition of oxidation of organic
substrates: styrene [4], linoleic acid, methyl or ethyl
linoleate [5], canola oil [6]. The oxygen uptake measure-
ment is the most direct method used to measure the
reaction extent. Moreover, the induced oxidation of methyl
linoleate and its radical long chain reaction is well
documented and well known to be inhibited by various
antioxidants [7,8].
Pan et al. [9] have examined the radical scavenging
effect of organosolv lignins from hybrid poplar and the
relationship between the antioxidant capacity, lignin struc-
ture and extraction conditions. The applicability of lignins
from different sources as antioxidants has been also suc-
cessfully tested [10]. Recently Garcia et al. [11] have
studied the effect of pre-treatment processes on the antio-
xidant capacity of miscanthus sinensis lignins.
Miscanthus x giganteus (MxG) is one of the biomass
resources which has attracted considerable attention as a
possible dedicated energy crop [12,13]. Indeed, MxG
*Corresponding author.

Effect of the Pre-Treatment Severity on the Antioxidant Properties of Ethanol Organosolv Miscanthus x giganteus Lignin 30
presents some valuable advantages: it is a perennial grass
which requires little nitrogen fertilizer or herbicide, it can
grow to over 3 m tall per year to produce from 20 to 25
tons of dry matter per hectare, and it is non invasive.
Moreover, it is a rhizomatous C4 grass species, which
has a high carbon dioxide fixation rate. These properties
make miscanthus an interesting raw material for industrial
bioconversion processes.
In the present work ethanol organosolv lignins were
extracted from Miscanthus x giganteus over a chosen
range of severity. The influence of the treatment severity
on the antioxidant capacity was studied using the oxygen
uptake index method in presence of methyl linoleate.
2. Experimental
2.1. Materials
The raw Miscanthus x giganteus (MxG) was harvested in
spring 2008 in Trier (Germany). The air-dried MxG was
milled to a particle size of 1 - 3 mm using a Wiley mill
and stored at room temperature during the course of this
study. The untreated feedstock contained 25% Klason
lignin, 37% cellulose and 36% hemicelluloses [12]. All
chemical reagents used in this study were purchased from
Sigma Aldrich and VWR (France) and used as received.
2.2. Ethanol Organosolv Lignin (EOL)
Extraction
25 g (dry weight, dry matter content about 90%) of
Miscanthus was treated with aqueous ethanol (EtOH/ H2O
= 0.5 - 0.65) in presence of sulfuric acid as a catalyst. The
solid to liquid ratio used was 1:8. The pre-treatments were
carried out in a 1.0 L glasslined pressure Parr reactor
equipped with a 4842 temperature controller (Parr
Instrument Company, Moline, IL). The pre-treated Mis-
canthus was washed with warm (60˚C) ethanol/water (8:1,
3 × 50.00 mL). The washes were combined and 3
volumes of water were added to precipitate the Ethanol
Organosolv Lignin, which was collected by centrifuga-
tion and air dried. EOL characterizations were previously
described [14,15].
2.3. Evaluation of Lignin Antioxidant Properties
Antioxidant properties of MxG organosolv lignins were
investigated by evaluating oxygen uptake inhibition dur-
ing oxidation of methyl linoleate. The induced oxida-
tion by molecular oxygen was performed in a gas-tight
borosilicate glass apparatus [5,16]. Butan-1-ol was used
as solvent for lignin dissolution. Temperature was set to
60˚C, initial conditions inside the vessel were as follows;
methyl linoleate (Fluka, 99%) concentration: 0.32 mol/L;
2,2’-azobisisobutyronitrile(AIBN) (Fluka, 98%) concen-
tration: 7.2 × 10–3 mol·L–1; lignin concentration: 0.2
g·L–1; oxygen pressure: 145 Torr. Oxygen uptake was
monitored continuously by a pressure transducer (Viatron
model 104). Without any additive, oxygen uptake is
roughly linear and constitutes the control. In the presence
of an antioxidant, oxygen consumption is slower, and we
estimated the antioxidative capacity of extract by com-
paring oxygen uptake at a chosen time (4 h), in the pres-
ence of this compound (pressure variation ΔPsample)
and in the absence of the compound (ΔPcontrol) accord-
ing to:
OUI = (ΔPcontrol – ΔPsample)/ΔPcontrol
This ratio defines antioxidative capacity as an oxygen
uptake inhibition index (OUI); it should spread from 0 to
100%, for poor and strong antioxidants, respectively, and
may be negative for proxidants. Do not add any kind of
pagination anywhere in the paper.
2.4. Error Analysis
For all EOL samples, oxygen update index values were
calculated from 2 independent experiments performed
under the same conditions. Error values (standard devia-
tion) were also estimated following the method described
by Mounanga et al. 2008 [16] and are about ~5% for all
the essays.
3. Results and Discussion
In recent work we have successfully developed an etha-
nol organosolv process for the pretreatment of MxG [12]
and we have confirmed that this process leads us to pro-
duce little degraded and relatively pure ethanol or-
ganosolv lignin (EOL) from miscanthus [14]. The isolation
of EOL fractions from MxG is illustrated in Figure 1.
Lignin was extracted using various experimental condi-
tions (Table 1), a temperature range between 170˚C and
190˚C, sulfuric acid (SA) concentration between 0.5%
Miscanthus x Giganteus
Organosolv treatment
t = 170˚C, 180˚C,190˚C
SA = 0.5%, 1.0%, 1.2%,1.6%
EtOH/H
2
O = 0.5, 0.65
t = 60 min
Black liquor
Filtration
Liquid
Phase
MxG
lignin
+H
2
O
Figure 1. Schematic of the or ganosolv process.

Effect of the Pre-Treatment Severity on the Antioxidant Properties of Ethanol Organosolv Miscanthus x giganteus Lignin 31
Table 1. Lignin oxygen uptake index, phenolic content, molecular weight and polydispersity values.
Experiment T (˚C) t (min) EtOH/ H2O (v/v) SA1 (%) CS2OUI3 (%)Phenolic OH
(mmol·g–1)
Aliphatic OH
(mmol·g–1) Mw (×103)Ip
EOL1 170 60 0.65 0.5 1.7558 2.34 3.11 6.5 2.6
EOL2 170 60 0.65 1.0 2.0862 2.72 1.78 6 1.6
EOL3 190 60 0.65 1.2 2.8686 4.04 1.07 3.6 1.4
EOL4 190 60 0.50 1.6 2.9391 3.93 1.26 3.2 1.4
1SA = Sulfuric Acid; 2CS = Combined Severity; 3OUI = Oxygen Uptake Index.
0 1 2 3 4 5 6
Time
[
h
]
40
35
30
25
20
15
10
5
0
Control
Oxygen uptake (hpa)
EOL1
EOL2
EOL3
EOL4
and 1.6%, and ethanol concentration of 50% or 65%, and
a reaction time of one hour. The investigation covered a
range of combined severity [(CS) = Log((t exp(T - Tref))-
pH] of 1.75 - 2.93; CS describes the severity of the pre-
treatment as a function of treatment time (t = min), tem-
perature (T˚C) and the pH of the medium[17]. The effect
of severity conditions on EOL chemical structure was
investigated and published recently [15]. The effect of
the treatment severity on miscanthus lignin structure has
been also studied and it was demonstrated that the in-
creasing in severity of the organosolv treatment was ac-
companied by (1) a decrease in the aliphatic OH groups,
(2) an increase of phenolic OH groups, (3) a decrease of
the weight average (Mw) molecular weight and polydis-
persity and (4) a strong increase in the degree of condensa-
tion and a cleavage of α or β aryl ether bonds [15].
The study of the antioxidant capacity of EOLs was re-
alized using the induced oxidation method of methyl
linoleate. Figure 2 shows the autoxidation of methyl
linoleate induced by AIBN-Azobisisobutyronitrile in ab-
sence (control) and in presence of lignin (EOL1, EOL2,
EOL3, EOL4) extracted at different pre-treatment sever-
ities. Antioxidant essays were realized under the same
conditions in presence of same lignin concentration (0.2
g·L–1). It appears that the autoxidation of methyl li-
noleate alone is almost linear [Figure 2 (control)]. By
comparing the obtained values in methyl linoleate oxi-
dation, it appears that lignin samples exhibited antioxi-
dant activity by slowing down oxidation of linoleate.
This behavior indicates that organosolv lignin acts as a
potential anti-oxidant that inhibits the oxidation of
methyl linoleate.
Figure 2. Oxygen uptake during the autoxidation of methyl
linoleate induced by AIBN in the presence of MxG organo-
solv lignins.
86% and EOL4, OUI = 91%) seem to be more efficient
than catechin (OUI = 76%). As catechin is a very effi-
cient antioxidant in this system [18], more efficient than
most of wood extracts, EOLs are very efficient antioxi-
dants.
Several comparisons were performed in order to under-
stand differences of lignin antioxidative capacities and to
examine links with lignin structures and experimental con-
dition of processes. Data of combined severity (CS), total
phenolic hydroxyl content, total aliphatic hydroxyl con-
tent determined by 31P NMR, average molecular weight
(Mw) and polydispersity (Ip) determined by GPC [15]
are also compiled in Table 1. Antioxidative capacities [OUI (%)] defined as the ratio
of oxygen uptakes at 4 h in presence of lignin are also
reported in Table 1. As can be seen in this table, results
of OUI are not the same for each lignin. These values
lead us to classify the antioxidant properties of EOLs
from the lowest (OUI = 58%) to the highest (OUI = 91%)
as follow EOL1 < EOL2 < EOL3 < EOL4. Furthermore
the oxidation of methyl linoleate in presence of a flavon-
oid polyphenolic antioxidant (catechin) was performed in
the same conditions. As a result, miscanthus organosolv
lignins recovered at high severity level (EOL3, OUI =
Figure 3 shows the Oxygen uptake index (%) as a
function of the combined severity. It can be observed that
OUI is positively correlated with the combined severity;
the correlation coefficient (R2) is about 0.98. The anti-
oxidant activity increases with the combined severity of
the organosolv pretreatment process. Thus the effect of
lignin extraction process on its antioxidant properties is
confirmed [9-19]. Lignins extracted at elevated tempera-
ture, longer reaction time, increased catalyst amount, and
dilute ethanol showed higher antioxidant activity.

Effect of the Pre-Treatment Severity on the Antioxidant Properties of Ethanol Organosolv Miscanthus x giganteus Lignin 32
1.5 2 2.5 3
Combined severity
100
90
80
70
60
50
y = 31x
R
2
= 0.9759
Oxygen uptake index (%)
Figure 3. Oxygen uptake index as a function of organosolv
pre-treatment combined severity.
The antioxidant efficiency of isolated lignin fractions
is described to be related to their structure, purity and
polydispersity [3,9,20]. Figures 4(a) and (b) show the
oxygen uptake index as a function of phenolic hydroxyl
and aliphatic hydroxyl content respectively. As we pre-
viously demonstrated, aliphatic hydroxyl moieties in
MxG organosolv lignin decreased with the severity of the
treatment while phenolic hydroxyl moieties increased
[15]. In our case it can be observed in Figures 4(a) and
(b) that OUI is correlated to phenolic hydroxyl group
(ArOH) and to aliphatic hydroxyl group (AlkOH) re-
spectively. OUI increases with the lignin phenolic OH
content and with the reduction of aliphatic OH. It is clear
from Figures 4(c) and (d) that lignin with high molecu-
lar weight and polydispersity had low antioxidant prop-
erties. Low molecular weights result from cleavage of
some inter-unit bonds in lignin and this degradation is
accompanied by an increasing of OH aliphatic content
and decreasing in OH phenolic group.
The radical scavenging activity of lignin phenolic com-
pounds depends not only on the hydrogen atom withdraw-
ing but also on stability of the radical formed [9]. Figure 5
shows the mechanism generally invoked for induced
oxidation of polyunsaturated acids [7-21]. It was also
reported that methoxyl groups ameliorate the antioxidant
activity [3-9]. In our recent published work results [15],
we have shown using 13C NMR that EOLs contain
methoxyl group (about 1.4 MeO per aromatic unit) and
that no demethoxylation was observed during the or-
ganosolv treatment, even at high severity. So the in-
creasing in the antioxidant activity observed in this study
could be rationalized by a simultaneous effect: 1) in-
creasing donating hydrogen atom and phenoxyl radical
formation due to higher phenolic group contents and 2)
stability of the radicals formed in presence of methoxy
group in ortho positions (Figure 5).
1.5 2 2.5 3 3.5 4 4.5
Phenolic OH (mmol·g
–1
)
100
90
80
70
60
50
y = 1.9741x2 + 6.3787x+31.44
R2 = 0.9635
OUI (%)
(a)
0.5 1 1.5 2 2.5 3 3.5
Aliphatic OH (mmol·g
–1
)
100
90
80
70
60
50
y = 15.096x2 – 76.826x+150.92
R
2
= 0.97
OUI (%)
(b)
3 3.5 4 4.5 5 5.5 6 6.5 7
Average molecular weight (Mw × 103)
100
90
80
70
60
50
y = –9.994x + 122.47
R
2
= 0.9988
OUI (%)
(c)
1 1.5 2 2.5 3
Polydispersity (Ip)
100
90
80
70
60
50
OUI (%)
(d)
Figure 4. Oxygen uptake index as a function of EOL pheno-
lic OH and aliphatic OH content, average molecular we ight
and polydispersity.

Effect of the Pre-Treatment Severity on the Antioxidant Properties of Ethanol Organosolv Miscanthus x giganteus Lignin 33
Products
Coupling reactions
MeO
LO
2
H+
LO
2
OH
OMe
MeO OMe
O.
LO
2
+ LO
2
LO
2
+ Products
LOOH + LO
2
AIBN + LH + O
2
LO
2
+ LH + O
2
Figure 5. Mechanism for induced oxidation of polyunsa-
turated aci ds.
4. Conclusion
In this work it has been shown that Miscanthus x giganteus
organosolv lignin have a potential application as antioxi-
dant. Lignin extraction process parameters are important
factors that can influence the antioxidant properties. In
this way, the obtained results of oxygen updated index
have confirmed that lignin antioxidant activity increased
positively with the severity treatment. Lignin fractions
extracted at elevated temperature and high catalyst con-
tents have revealed a better antioxidant behavior than
catechin a well known antioxidant. The present study has
also confirmed the close correlation between lignin che-
mical structure and its antioxidant activities; lignin frac-
tions with higher phenolic hydroxyl groups content, lower
molecular weight, polydispersity and aliphatic hydroxyl
groups content gave higher values of antioxidant activity.
5. Acknowledgements
The authors gratefully acknowledge the financial support
of the CPER 2007-2013 “Structuration du Pôle de Compé-
titivité Fibres Grand’Est” (Competitiveness Fibre Cluster),
through regional (Région Lorraine), national (DRRT and
FNADT) and European (FEDER) funds.
REFERENCES
[1] X. B. Zhao, K. K. Cheng and D. H. Liu, “Organosolv
Pretreatment of Lignocellulosic Biomass for Enzymatic
Hydrolysis,” Applied Microbiology and Biotechnology,
Vol. 82, No. 5, 2009, pp. 815-827.
doi:10.1007/s00253-009-1883-1
[2] A. Ragauskas, C. K. Williams, B. H. Davison, G. Bri-
tovsek, J. Cairney, C. A. Eckert, W. J. Frederick Jr., J. P.
Hallett, D. J. Leak, C. L. Liotta, J. R. Mielenz, R. Murphy,
R. Templer and T. Tschaplinski, “The Path Forward for
Biofuels and Biomaterials,” Science, Vol. 311, No. 5760,
2006, pp. 484-489. doi:10.1126/science.1114736
[3] T. Dizhbite, G. Telysheva, V. Jurkjane and U. Viesturs,
“Characterization of the Radical Scavenging Activity of
Lignins-Natural Antioxidants,” Bioresource Technology,
Vol. 95, No. 3, 2004, pp. 309-317.
doi:10.1016/j.biortech.2004.02.024
[4] G. W. Burton, T. Doba, E. Gabe, L. Hughes, F. L. Lee, L.
Prasad and K. U. Ingold, “Autoxidation of Biological
Molecules. 4. Maximizing the Antioxidant Activity of
Phenols,” Journal of the American Chemical Society, Vol.
107, No. 24, 1985, pp. 7053-7065.
[5] H. Eloualja, D. Perrin and R. Martin, “Kinetic Study of
the Thermal Oxidation of All-Trans-β-Carotène and Evi-
dence of Its Antioxigen Properties,” New Journal of
Chemistry, Vol. 19, No. 11, 1995, pp. 863-872.
[6] U. N. Wanasundara and F. Shahidi, “Stabilization of Ca-
nola Oil with Flavonoids,” Food Chemistry, Vol. 50, No.
4, 1994, pp. 393-396. doi:10.1016/0308-8146(94)90211-9
[7] C. Rousseau, C. Richard and R. Martin, “Influence
Inhibitrice Comparé des Vitamines C et E sur l’Oxydation
Induite du Linoléate de Methyl vers 80˚C et Effet de
Synergie,” Chimie Physique, Vol. 80, No. 11-12, 1983, pp.
827-829.
[8] N. Uri, “Physico-Chemical Aspects of Autoxidation,” In:
W. O. Lundberg, Ed., Autoxidation and Antioxidants, In-
ter-Science Publishers, New York, 1961.
[9] X. J. Pan, J. F. Kadla, K. Ehara, N. Gilkes and J. N. Sad-
dler, “Organosolv Ethanol Lignin from Hybrid Poplar as
a Radical Scavenger: Relationship between Lignin Struc-
ture Extraction Conditions and Antioxidant Activity,”
Journal of Agriculture and Food Chemistry, Vol. 54, No.
16, 2006, pp. 5806-5813. doi:10.1021/jf0605392
[10] V. Urgatondo, M. Mitjans and M. P. Vinardell, “Applica-
bility of Lignins from Different Sources as Antioxidants
Based on the Protective Effects on Lipid Peroxidation
Induced by Oxygen Radicals,” Industrial Crops and
Products, Vol. 30, No. 2, 2009, pp. 184-187.
doi:10.1016/j.indcrop.2009.03.001
[11] A. Garcia, A. Toledano, M. A. Andres and J. Labidi,
“Study of the Antioxidant Capacity of Miscanthus Sinen-
sis Lignins,” Process Biochemistry, Vol. 45, No. 6, 2010,
pp. 935-940. doi:10.1016/j.procbio.2010.02.015
[12] N. Brosse, P. Sannigrahi and A. Ragauskas, “Pretreat-
ment of Miscanthus x giganteus Using the Ethanol Or-
ganosolv Process for Ethanol Production,” Industrial &
Engineering Chemistry Research, Vol. 48, No. 18, 2009,
pp. 8328-8334. doi:10.1021/ie9006672
[13] A. Sørensen, P. J. Teller, T. Hilstrom and B. K. Ahring,
“Hydrolysis of Miscanthus for Bioethanol Production Us-
ing Dilute Acid Presoaking Combined with Wet Explosion
Pre-Treatment and Enzymatic Treatment,” Bioresource
Technology, Vol. 99, No. 14, 2008, pp. 6602-6607.
doi:10.1016/j.biortech.2007.09.091
[14] R. El. Hage, N. Brosse, L. Chrusciel, C. Sanchez, P. San-
nigrahi and A. Ragauskas, “Characterization of Milled
Wood Lignin and Ethanol Organosolv Lignin from Mis-
canthus,” Polymer Degradation and Stability, Vol. 94, No.

Effect of the Pre-Treatment Severity on the Antioxidant Properties of Ethanol Organosolv Miscanthus x giganteus Lignin 34
10, 2009, pp. 1632-1638.
doi:10.1016/j.polymdegradstab.2009.07.007
[15] R. El Hage, N. Brosse, P. Sannigrahi and A. Ragauskas,
“Effect of Process on the Chemical Structure of Miscan-
thus Ethanol Organosolv Lignin,” Polymer Degradation
and Stability, Vol. 95, No. 6, 2010, pp. 997-1003.
doi:10.1016/j.polymdegradstab.2010.03.012
[16] T. K. Mounanga, P. Gérardin, B. Poaty, D. Perrin and C.
Gérardin, “Synthesis and Properties of Antioxidant Am-
phiphilic Ascorbate Salts,” Colloids and Surfaces A:
Physicochemical Engineering Aspects, Vol. 318, No. 1-3,
2008, pp. 134-140. doi:10.1016/j.colsurfa.2007.12.048
[17] D. Montane, X. Farriol, J. Salvado, P. Jollez and E. Chor-
net, “Fractionation of Wheat Straw by Steam-Explosion
Pre-Treatment and Alkali Delignification. Cellulose Pulp
and Byproducts from Hemicelluloses and Lignin,” Jour-
nal of Wood Chemistry and Technology, Vol. 18, No. 2,
1998, pp. 171-191. doi:10.1080/02773819809349575
[18] P.-N. Diouf, A. Merlin and D. Perrin, “Antioxidant Prop-
erties of Wood Extracts and Colour Stability of Woods,”
Annals of Forest Science, Vol. 63, No. 5, 2006, pp. 525-534.
doi:10.1051/forest:2006035
[19] H. Nadji, P.-N. Diouf, A. Benaboura, Y. Bedard, B. Riedl
and T. Stevanovic, “Comparative Study of Lignins Iso-
lated from Alfa Grass (Stipa tenacissima L.),” Biorsource
Technology, Vol. 100, No. 14, 2009, pp. 3585-3592.
doi:10.1016/j.biortech.2009.01.074
[20] C. Pouteau, P. Dole, B. Cathala, L. Averous and N. Bo-
quillon, “Antioxidant Properties of Lignin in Polypropyl-
ene,” Polymer Degradation and Stability, Vol. 81, No. 1,
2003, pp. 9-18. doi:10.1016/S0141-3910(03)00057-0
[21] E. Niki, T. Saito, A. Kawakami and Y. Kamiya, “Inhibi-
tion of Oxidation of Methyl Linoleate in Solution by Vi-
tamin E and Vitamin C,” The Journal of Biological
Chemistry, Vol. 259, No. 7, 1984, pp. 4177-4182.