trends in analytical chemistry, vol. 15, no.
2, 1996
Experimental design
II. Optimization
Pedro W. Araujo, Richard
G. Brereton *
Bristol, UK
This article is concerned with improving the
output in an analytical system, as a function
of several experimental factors, in order to
obtain the optimum operational condition.
Topics covered are sequential and simulta-
neous designs. The former include simplex
and steepest ascent designs and the latter
star, central composite, face centred cube,
rotatable central composite, Doehlert and
mixture designs. Advantages and disadvan-
tages when they are applied are also
discussed.
1. Introduction
The term optimization has been used in chem-
istry for a long time, and for this reason it is a word
that always has significance in this branch of the
science. The general goal in any optimization is to
discover the conditions that produce the best out-
put. This task is usually performed by monitoring
the influence of one factor at a time on an experi-
mental response. The main disadvantages of one
factor optimization is that it is time consuming and
that there is a risk of misinterpreting the results if
important interactions between factors are present.
Important ideas have been generated in chemical
optimization with the arrival of chemometrics and
among them the most relevant are sequential and
simultaneous designs in analytical optimization.
The major difference between these groups is that
in the former it is necessary to analyse the data via
computational curve fitting subsequent to the
experiments, whereas in the latter the answer is
obtained experimentally.
* Corresponding author.
0 1996 Elsevier Science R V. All riehtr reserved
63
2. Optimization
2.1. Sequential designs
When the experimenter has only limited knowl-
edge about how far the optimum region is from the
starting experimental point, sequential designs are
useful in order to obtain guidance and direction for
future experimentation. There are several kinds of
sequential designs, the most notable being the sim-
plex design and modified versions, the steepest
ascent design and the evolutionary optimization
design [ l-51.
A simplex is defined as the simplest possible IZ-
dimensional figure. In one dimension is a line, in
two dimensions a triangle, in three dimensions a
tetrahedron and in more dimensions a hyper-
triangle.
There is a great deal of literature on the subject
of simplex optimization. The construction of this
kind of design is based on the following rules:
Choose the number of factors (N) and set N+ 1
experimental starting points or vertices, e.g., for
2 factor (xl andx,) we will have 3 vertices which
can be represented as a triangle ABC (Fig. 1a).
Define the experimental space, especially coding
of factors so that each axis is equally significant.
For example, if one factor is temperature (e.g.,
range 20-80°C) and the other pH (e.g., 1-5) a
change of one pH unit is far more significant than
a change of 1°C. Hence factors must be coded
appropriately.
Perform the experiments under the conditions
represented by each vertex and rank the experi-
mental responses, e.g., for 2 factors and 3 verti-
ces, 3 responses can be determined ranked for 1
(best) to 3 (worst) corresponding to the
responses at the vertices C =93, B =79 and
A = 64, respectively, in Fig. 1b.
Eliminate the condition that has produced the
worst response (vertex A in Fig. 1b).
Determine the mid-point (p) between vertices C
and B and reflect the eliminated vertex through
the mid-point to generate a new vertex, e.g., the
vertex A in Fig. lc has been reflected through
the mid-pointp to obtain the new vertex D, which
provides conditions for the next experiments.
64 trends in analytical cherr?isWy,vol. 15, no. 2, 7996

a) x1
D

B B=79

D
4
C

x2
I,
A=64
c=93

x2

Fig. 1. Basic simplex for two factors: (a) initial simplex; (b) simulated responses; and (c) reflection of the worst
vertex to obtain the new condition.

l If the experimental conditions provided by D
produce a worst response again, reject the next
to the worst vertex (B in our example).
Sometimes when the new vertices are calculated,
the values obtained lie outside the allowable exper-
imental domain, for instance, the new volume
obtained is higher than the capacity of the appara-
tus. In this case, the simplex should be contracted
and a new vertex must be generated.
An illustrative example based in the rules
described above can be seen in Table 1 and Fig. 2,
which show the progress of a simplex design in the
maximization of a hypothetical response. Two fac-
tors have been considered which are described in
the vertices ( Vi) of a triangle (N+ 1 = 3). For a
better understanding, the variation of the factors is
presented in coded levels (from - 1 to + 1) and
the response is expressed as a percentage in area.
Table 1 shows the initial simplex ( V, V,V,) and the
simplex progress followed in this example. Follow-
ing the algorithm described, responses at vertices
V, to V, were obtained and V, was rejected because
Table 1
Progress in a simplex design in the maximization
imental response when 2 factors are varied
its response was the worst, and so V, is obtained
reflecting VI through the mid-point between V, and
V,. This new experiment is run and V, is rejected
and a new vertex V, is found. Again, the new exper-
imental point is run and so until reaching some
experimental condition which produce the opti-
mum response. The best response in this example
was Vlo. An important aspect of the simplex
designs is to know when the procedure has finished.
There are many different stopping rules [ 6-91, but
sometimes the best method involves the experi-
menter’s skill in judging for him/herself whether
a true optimum has been found [ 61.
The most notable disadvantages of the simplex
designs are that the optimum is confined to a local
point instead of the global region, often a large
number of experiments is required, replicate infor-
mation is not obtained, there is no information
about how confident we are in the model and with
L
0
of an exper- k 0.6
‘c

Vertex x1 X2 Response (%) z 0.2

r,
0
1 -1.0 - 1 .o 35
2 -0.6 - 1 .o 39 2 -0.2
3 -0.8 -0.6 58 :
Q)
4 -0.4 -0.6 60
5 -0.6 -0.2 64 $’ -0.6
6 -0.2 -0.2 79 u
7 -0.4 0.2 82 u”
8 0.0 0.2 93 -1.0 d
9 -0.2 0.6 94
10 0.2 0.6 99 -1.0 -0.6 -0.2 0.2 0.6 1.0
11 0.4 0.2 95
12 0.6 0.6 96 Coded levels of the factor xl
13 0.4 1.0 97
Fig. 2. Progress in a simplex design for two factors.
trends in analytical chemistry, vol. 15, no. 2, 1996
an inappropriate choice of step size and starting
conditions it is possible to miss the optimum
completely.
The steepest ascent (or descent) method has also
been proposed as an approach to finding an opti-
mum [ 4,10,1 11. The main principle is to use local
response surface modelling to determine the gra-
dient of an overall design. Above, designs were
performed over the entire experimental region
which then produced a mathematical model for the
optimum. Steepest ascent methods starts over a
small region rather like the simplex approach. For
two factors this nominally involves performing 4
experiments arranged as a 2 level factorial design
to give a response of the form presented in Eq. 1
of Part I of this article. Taking derivatives, it is
possible to determine the gradient over each factor
and setting dv/Jx,, and ;?Y/&x~at 0, to determine
the direction of maximum change. There has been
some discussion in the literature as to precisely how
to estimate this gradient [ 121. Once the gradient is
determined, new experiments are performed cen-
tred on
x:,,, = GM + aAx
where a is the step size and Ax is obtained from
the gradient. This is continued until an optimum is
reached.
The main disadvantage to the analytical chemist
is that if more than 2 factors are significant, the
number of experiments per estimate increases by 2
for each factor. Hence a 4 factor process requires
16 experiments each time. This is prohibitively
large, so this approach is not popular.
2.2. Simultaneous designs
The basic idea in simultaneous designs is to rec-
ord one or more selected experimental responses
for a set of experiments carried out in a systematic
way in order to predict the optimum and the inter-
action effects using regression analysis generally.
Factorial designs at 2 levels are suitable in the
estimation of first-order and interaction effects, but
they fail when additional effects such as second-
order effects are significant. For optimization some
second order effects are necessary.
Star designs provide a simple way to fit a model
described by the following equation:
65
Table 2
Stardesignforthreefactors
No. Xl X2 X3
1 0 0 0
2 -1 0 0
3 +1 0 0
4 0 -1 0
5 0 +1 0
6 0 0 -1
7 0 0 +1
where !I,,,&, is the second-order term. The follow-
ing rules can help to construct a star design.
0 Choose the number of factors to be studied (N),
e.g., 3 factors.
Establish the number of experiments to be done
according to the expression 2N+ 1, e.g., for 3
factors it will be necessary to perform 7
experiments.
Locate a central experimental point and a step
size, in the space of the coded variables, from
which other factor combinations are generated
by moving the same positive and negative dis-
(I)
tance ( = step size), e.g., for 3 factors the exper-
imental arrangement is shown in Table 2, where
the centre corresponds to the point (0, 0, 0).
The experiments performed according to Table 2
are exactly sufficient to estimate 2N+ 1 para-
meters of the model, e.g., for 3 factors we expect
7 parameters, bO, b,, bZ, b,, b, , , bz2 and b,,.
The main disadvantage of these designs is that
they do not allow for models involving interac-
tions. This is evident from Table 2, where the mul-
tiplication of any column by another in order to
obtain the interaction factor will produce a result
of zero in all the experimental points. This lack of
estimation effects of interaction can cause the
model to fail if they have significance. Also, there
are no degrees of freedom to estimate how well the
model is obeyed. To overcome this problem, a bet-
ter approximation to a multifactor space model has
been developed [ 131. Belonging to this approach
are the central composite designs, the face-centred
cube designs, rotatable central composite designs,
Doehlert designs and mixture designs. For N fac-
tors we can allow an extension of the model to
j=b,,+b,x] +b2x2 +b3x3+...+b,,++bIZx,x2
+ b,,x,x, + bmxzx3 + .. . + b,,- I ,hrx,v- IAN
+ b, ,x: + b2?x; + b33x; + . ..b.,+; (3)
66 trends in analytical chemistry, vol. 15, no. 2, 7996

Table 3 Table 4
Central composite designforthreefactors Face-centredcubedesignforthreefactors

No. Xl X2 X3 No. Xi X2 X3

1 0 0 0 1 0 0 0
2 -1 0 0 2 -1 0 0
3 +1 0 0 3 +1 0 0
4 0 -1 0 4 0 -1 0
5 0 +1 0 5 0 +1 0
6 0 0 -1 6 0 0 -1
7 0 0 +1 7 0 0 +1
8 -0.5 -0.5 -0.5 8 -1 -1 -1
9 -0.5 +0.5 -0.5 9 -1 i-1 -1
10 +0.5 +0.5 -0.5 10 +1 +1 -1
11 +0.5 -0.5 -0.5 11 t-1 -1 -1
12 -0.5 -0.5 +0.5 12 -1 -1 +I
13 -0.5 +0.5 +0.5 13 -1 +1 +1
14 i-o.5 +0.5 +0.5 14 +1 +1 +1
15 +0.5 -0.5 +0.5 15 +1 -1 +1

Central composite designs are the result of
superimposing a factorial design at 2 levels ( 2N)
on a star design (2N+ 1). The rules followed in its
construction are the same as explained for both
kinds of design and the total number of factor com-
binations will be given by 2N + 2N+ 1 to define the
linear, quadratic and interaction terms of the model
together with an estimation of the error in the terms.
An experimental arrangement for 3 factors in a
central composite design is shown in Table 3,
where experiments 1 to 7 define the star and 8 to
15 define the factorial design. In this example the
distance from the centre to the negative and posi-
tive extreme in each design is different ( + 1 in the
star and f 0.5 in the factorial design), but when
the same positive and negative distance is taken
from the centre of the design it is called a face-
centred cube design. The arrangement of the exper-
imental points of this design is the same as for the
central composite except that the coded levels from
rotatable central composite design can be used.
Rotatable central composite designs are identical
with a faced-centred cube design except that the
points defined for the star design must be changed
to ( + [2N] “4 ,...,O) and those generated by the fac-
torial design remain unchanged. In this way the
design generates information equally in all direc-
tions, e.g., the variance of the estimated response
is the same at all points on a sphere centred at the
origin. This is why these designs are called rotat-
able, because a rotation of design about the origin
does not alter the variance contours [ 171.
Taking into account the above considerations
about rotatability in Table 4 all the levels + 1 in
experiments 8-l 5 must change to ) 1.68, whereas
experiments l-7 do not change (see Table 5). As
Table 5
Rotatablecentralcomposite
designforthreefactors
No. Xl x2 X3

the factorial part must be the same than the star part
1 0 0 0
changing the levels f 0.5 by + 1 (see Table 4 and
2 -1 0 0
compare with Table 3). This kind of design has 3 +1 0 0
been used in the optimization of new methods 4 0 -1 0
[ 14,151. 5 0 +1 0
Sometimes the centre in this kind of design is 6 0 0 -1
7 0 0 +1
replicated 5 or more times, because the design
8 -1.68 -1.68 -1.68
assumes that the pure experimental uncertainty is 9 -1.68 +1.68 -1.68
related to the distance from the centre of the design, 10 +I.68 +I.68 -1.68
but sometimes it is also a function of the distance 11 +1.68 -1.68 -1.68
from the other experimental points [ 161. To fulfil 12 -1.68 -1.68 +I.68
13 -1.68 +1.68 +1.68
the condition of dependence only on the distance
14 +1.68 +1.68 +1.68
from the centre of the design and independence of 15 +I.68 -1.68 +1.68
the location of the other experimental points, a
trends in analytical chemistry, vol. 15, no. 2, 1996 67

Table 6 Table 7
Rotatable central composite design and yields obtained in Experimental field in a Doehlert matrix for three factors
enamine synthesis by the TiCId method
No. Xl X2 X3
No. Xl X2 Yield (%)
1 0 0 0
1 -1 0 73.4 2 -1 0 0
2 +1 0 69.7 3 -0.5 - 0.866 0
3 -1 -1 88.7 4 -0.5 - 0.289 -0.816
4 +1 -1 98.7 5 1 0 0
5 - 1.41 +1 76.8 6 0.5 0.866 0
6 +1.41 +1 84.9 7 0.5 0.289 0.816
7 0 0 56.6 8 -0.5 0.866 0
8 0 0 81.3 9 -0.5 0.289 0.816
9 0 - 1.41 96.3 10 0 - 0.577 0.816
10 0 + 1.41 96.4 11 0.5 - 0.866 0
11 0 0 87.5 12 0.5 - 0.289 -0.816
12 0 0 96.1 13 0 0.577 -0.816
13 0 0 90.5

an example of this design we present the synthesis
of enamine from methyl isobutyl ketone and mor-
pholine by the TiCI4 method [ 181. This synthesis
was carried out considering the effect of two fac-
tors, the amount of TiCl,/ketone (x,) varied
between 0.50 and 1.OOmol and morpholine/ketone
(x2) varied between 3.00 and 8.00 mol. The exper-
imental design and yields obtained are shown in
Table 6, where the factors are given in coded levels.
The quadratic response surface model fitted to the
experiments was

ji = 93.46 + 2.22x, + 9.9~~

+ 3.42x,x2 -4.37x: - 10.32x; (4)

Eq. 4 shows that the interaction and the second-

Fig. 3. Doehlerl design generation for two factor. The
numbers correspond to the experimental points
investigated.
order polynomial terms have to be considered in
order to obtain a real optimum experimental con-
dition, which in this study was x1 =0.47 and
x2 = 0.56, both in coded levels. Further experiments
performed under these conditions afforded a quan-
titative yield of 98+2% and confirmed the opti-
mum (see Table 6).
The simultaneous designs described in this sec-
tion are useful for optimizing an analytical method
if the number of factors is not too large. The effi-
ciency, defined as the number of parameters to be
estimated divided by the number of factors com-
bined, is high for up to 5 factors but decreases
rapidly for 6 or more factors.
Another kind of simultaneous design is the uni-
form shell design developed by Doehlert [ 191,
which has the following characteristics:
0 A uniform distribution of the experimental
points is allocated on the surface of a hyper-
sphere, e.g., for two factors the generation of this
design (hexagonal shape) is presented in Fig. 3.
0 The number of experiments is given by
N*+ N+ 1. For instance, 3 factors will require
13 experiments organized according to Table 7.
68 trendsin analyticalchemistry,vol. 15, no. 2, 7996

I(1 II IO II
conditions: 2-S% (v/v) HCl, ashing temperature

.Q@y5&ls
4 3 4 3 4 3 I3 14
400-l 300°C and atomization temperature 2800°C.
The last type of design discussed in this section
is mixture designs. These designs are particularly
useful when the experimenter has to deal with pro-
portions which are restricted to some limits in the
Fig. 4. Extension of the experimental Doehlert matrix possible combination of the factors. For example,
to another experimental domain by using previous the proportion of three solvents A, B and C cannot
adjacents points. exceed lOO%, so if the proportion of A and B is
established then the proportion of C must be
0.212 0.200 cv NV lOO- (A+B)%.
0.065
0.346 ” 0.341
. \

0.217 0.27; 0.305

,
, .
(rf) o&3- -0$5
0.370

0.285 0.289

0.0 2.5 5

Fig. 5. Experimental results for the Doehlert matrix

given in Table 7 used in the optimization of some
factors that affect the MO signal. For HCI and ashing
temperature two scales are shown which correspond
to coded (CV) an natural (NV) variables. Atomization
I
temperature used: (thin line) 2763”C, (thick line) solvent A (%)
2600°C; and (dashed line) 2437°C.
Fig. 6. Experimental points in a mixture design for
three solvents.
l Each factor is analysed at different numbers of
levels. Table 7 shows how the factors x1, x2 and
xg are studied at 5, 7 and 3 levels, respectively.
This particular characteristic is relevant when
some factors are subject to some restrictions such
as cost or instrumental constraints, so their study
with a small number of levels is necessary.
l Extension of the experimental matrix to another
experimental domain is done by using previous
adjacents points (Fig. 4).
The optimization of some factors that affect the
MO signal by electrothermal AAS has been carried
out using a Doehlert matrix [20]. The factors
selected in this study were HCl concentration (x1)
varied between 0 and 10% (v/v), ashing temper-
ature (x2), varied between 400 and 22OO”C, and Fig. 7. Mixture design used in the optimization of sol-
atomization temperature (x3), varied between vent selectivity. Each experimental point given as a
2400 and 2800°C. The results shown in Fig. 5 dem- roman numeral corresponds to molar fractions given
onstrate that the best signals are obtained in the in parenthesesof (hexane + methylene chlo-
experimental region described by the following ride + acetonitrile + methyl tert.-butyl ether).
trends in analytical chemistry, vol. 15, no. 2, 7996 69

Table 8
Capacity factor results for seven mobile phases in Fig. 7

Solutes Points corresponding to Fig. 7

I II III IV V VI VII

2-OCH, 0.58 0.57 0.59 0.65 0.67 0.54 0.67

1-NO, 0.86 1.20 1.62 1.10 1.36 0.90 1.30
1,WXH,), 1.15 0.82 1 .oo 1.02 0.95 0.91 1.02
1,5-(NO,), 2.37 3.27 3.70 2.98 3.62 2.63 3.63
1-CHO 2.75 1.69 2.45 2.22 2.11 2.27 2.33
2X0&H, 3.29 2.49 2.83 3.00 2.99 2.78 3.25
1-CO,CH, 3.31 2.71 3.07 3.13 3.33 2.85 3.57
2-CHO 3.97 2.22 3.25 3.19 2.83 3.12 3.17
1-CH,CN 4.06 4.73 7.23 4.86 6.09 4.83 6.30
1-OH 4.44 8.17 6.65 6.77 7.14 6.27 8.00
1-COCH, 5.17 2.58 3.54 3.71 3.25 3.72 3.72
2-COCH, 7.33 3.33 4.76 5.14 4.39 5.16 5.10
2-OH 7.98 11.86 11.35 10.69 11.58 11.57 13.42

There are several types of mixtures designs one
of the most common being the simplex-lattice
design, which was introduced by Scheffi [ 2 1,221
and formed the base upon which the theory of
experimental design for mixtures was built. Some
rules can be considered in the building of this kind
of design:
l Choose the number of factors (N), e.g., three
solvents: acetone, water and methanol.
l Represent the composition of the N factors by a
simplex in N- 1 dimensional space, e.g., for the
three solvents considered above, this is a triangle,
as shown in Fig. 6.
l Select the experimental points to be studied as
2N-‘. These experimental points correspond to
N permutations of N pure components, N!/
[ 2!(N - 2) !] permutation of full binary
mixtures, N!l[3!(N -3)!] permutation of all
ternary mixtures, and so on, e.g., for 3 compo-
nents, 7 experiments have to be performed as
follows: 3 with just one component ( 100% pure
solvent), 3 with a binary mixture of 50% of each
component and 1 with an equimolar mixture of
33.33% of each component. These experimental
points are represented in Fig. 6 by the symbol the
shaded circles.
Mixture designs were used in the optimization
of solvent selectivity in liquid-solid chromatogra-
phy for separating a mixture of 13 substituted naph-
thalene [23]. The seven experimental points
studied are shown in Fig. 7 and they correspond to
changes in selectivity as a result of variation of the
mobile phase composition. Each experimental
point is a quaternary mixture of four standard sol-
vents and the responses measured were the sepa-
ration factor and the value of the capacity factor
between two adjacent bands which are shown in
Table 8. The conclusion reached in this work was
that none of the seven standard mobile phases alone
provides a complete separation of the sample com-
ponents, so the optimum solvent composition is
given as 0.83% acetonitrile, 0.67% methyl tert.-
butyl ether and 3.05% methylene chloride by vol-
ume in hexane.
In spite of the variety of other mixture designs,
they have not been exploited extensively in chem-
istry [6].
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Thermally assisted hydrolysis and alkylation
as a novel pyrolytic approach for the
structural characterization of natural
biopolymers and geomacromolecules
J.C. del Rio *, F. Martin,
F.J. Gonzalez-Vila
Seville, Spain
The high-temperature reaction of macromol-
ecules with tetra-alkylammonium hydroxides
has significantly improved the use of the con-
ventional pyrolytic procedures which are
widely used for the structural characteriza-
tion of natural biopolymers and geomacro-
molecules. This new pyrolytic approach has
overcome some of the analytical limitations
of conventional pyrolysis allowing the detec-
tion of polar compounds as their alkyl deriv-
atives. We report recent developments of the
high-temperature hydrolysis and alkylation
procedure, carried out in the presence of
tetra-alkylammonium hydroxides, for the
structural characterization of natural biopo-
lymers and geomacromolecules such as lig-
nins, humic substances, coals, asphaltenes
and kerogens.
0165-9936/96/$15.00
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Pedro W. Araujo and Richard G. Brereton are at
the School of Chemistry, University of Bristol,
Cantock’s Close, Bristol BS8 I TS, UK. For
bibliographical details see Part I of this article.
1. Introduction
Analytical pyrolysis coupled with mass spec-
trometry (Py-MS) or gas chromatography-mass
spectrometry ( Py-GC-MS) is now recognized as
a powerful analytical technique for the chemical
structural characterization of natural biopolymers
and geomacromolecules [ l-81. The polymeric
material undergoes heat-induced bond dissociation
to give lower-molecular-mass compounds which
reflect the composition of the original polymer.
Analytical pyrolysis has many well known
advantages.
In many instances it is very reproducible and the
results can be interpreted both qualitatively and
quantitatively.
No chemical pre-treatments and/or fractiona-
tions are necessary, thus avoiding possible con-
tamination from laboratory manipulations.
Predominantly primary pyrolysis products are
obtained.
* Corresponding author.
0 1996 Elsevier Science B.V. All rights reserved