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 MOLECULAR
CHARACTERIZATION
OF POLYMERS
A Fundamental Guide
 MOLECULAR
CHARACTERIZATION
OF POLYMERS
A Fundamental Guide

Edited by

MUHAMMAD IMRAN MALIK

H.E.J. Research Institute of Chemistry,
International Center for Chemical and Biological Sciences (ICCBS),
University of Karachi, Karachi, Pakistan

JIMMY MAYS
Department of Chemistry, University of Tennessee,
Knoxville, TN, United States

MUHAMMAD RAZA SHAH

H.E.J. Research Institute of Chemistry,
International Center for Chemical and Biological Sciences (ICCBS),
University of Karachi, Karachi, Pakistan
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 Contributors
Luca Baiamonte Department of Chemistry,
Colorado School of Mines, Golden, CO, United
States
Josef Brandt Department Anaylsis, Leibniz-
Institut f€
ur Polymerforschung Dresden e.V.,
Dresden, Germany
Taihyun Chang Department of Chemistry and
Division of Advanced Materials Science,
Pohang University of Science and Technology,
Pohang, South Korea
Mark D. Dadmun Department of Chemistry,
University of Tennessee, Knoxville; Chemical
Sciences Division, Oak Ridge National
Laboratory, Oak Ridge, TN, United States
Nadia Edwin Department of Health and
Biomedical Sciences, AdventHealth University,
Orlando, FL, United States
Anthony P. Gies The Dow Chemical Company,
Lake Jackson, TX, United States
David Gillespie Tosoh Bioscience LLC, King of
Prussia, PA, United States
Andrew Gorman School of Materials Science &
Engineering, Georgia Institute of Technology,
Atlanta, GA, United States
Alexander S. Gubarev Department of
Molecular Biophysics and Physics of Polymers,
Saint-Petersburg State University, Saint-
Petersburg, Russia
Shaw Ling Hsu Polymer Science and
Engineering Department, University of
Massachusetts (Amherst), Amherst, MA,
United States
Wayne Huberty Advanced Composites
Institute, Mississippi State University,
Starkville, MS, United States
Muhammad Imran H.E.J. Research Institute of
Chemistry, International Center for Chemical
ix
and Biological Sciences (ICCBS), University of
Karachi, Karachi, Pakistan
S. Kim Ratanathanwongs Williams Department
of Chemistry, Colorado School of Mines,
Golden, CO, United States
Albena Lederer Department Anaylsis, Leibniz-
Institut f€
ur Polymerforschung Dresden e.V.;
School of Science, Technische Universit€
at
Dresden, Dresden, Germany; Department of
Chemistry and Polymer Science, Stellenbosch
University, Matieland, South Africa
Wei Lu Tosoh Bioscience LLC, King of Prussia,
PA, United States
Muhammad Imran Malik H.E.J. Research
Institute of Chemistry, International Center for
Chemical and Biological Sciences (ICCBS),
University of Karachi, Karachi, Pakistan
Jimmy Mays Department of Chemistry,
University of Tennessee, Knoxville, TN, United
States
Toshikazu Miyoshi Department of Polymer
Science, The University of Akron, Akron, OH,
United States
Harald Pasch Department of Chemistry and
Polymer Science, University of Stellenbosch,
Stellenbosch, South Africa
Jigneshkumar Patel Intel Corporation
(Chandler), Chandler, AZ, United States
Georges M. Pavlov Institute of Macromolecular
Compounds, Russian Academy of Sciences,
Saint-Petersburg, Russia
Igor Perevyazko Department of Molecular
Biophysics and Physics of Polymers, Saint-
Petersburg State University, Saint-Petersburg,
Russia
Jawadur Rehman H.E.J. Research Institute of
Chemistry, International Center for Chemical
x Contributors
and Biological Sciences (ICCBS), University of
Karachi, Karachi, Pakistan
Sebastien Rouzeau Tosoh Bioscience LLC, King
of Prussia, PA, United States
Paul S. Russo School of Materials Science &
Engineering; School of Chemistry &
Biochemistry, Georgia Institute of Technology,
Atlanta, GA, United States
Salim Saifullah H.E.J. Research Institute of
Chemistry, International Center for Chemical
and Biological Sciences (ICCBS), University of
Karachi, Karachi, Pakistan
Muhammad Raza Shah H.E.J. Research
Institute of Chemistry, International Center for
Chemical and Biological Sciences (ICCBS),
University of Karachi, Karachi, Pakistan
William C. Smith Department of Chemistry,
Colorado School of Mines, Golden, CO, United
States
Ali Soleymannezhad Tosoh Bioscience LLC,
King of Prussia, PA, United States
Kiril A. Streletzky Department of Physics,
Cleveland State University, Cleveland, OH,
United States
Michael Toney Department of Chemistry,
Colorado School of Mines, Golden, CO, United
States
Xujun Zhang Wyatt Technology Corporation,
Santa Barbara, CA, United States
Weiwei Zhao Ningbo Materials Institute,
Ningbo, China
 Foreword

As we contemplate the centennial of macromolecular chemistry, we may be reasonably

certain that Hermann Staudinger never said “Polymer scientia est omnis divisa in partes tres,”
but if he had, he would have been right! The first part of polymer chemistry is synthesis:
what molecular structures can we make, how easily, and how reproducibly? The third part
comprises material properties: what is this polymer good for, how can we make it better, and
how can we combine multiple desirable properties in one material? The intermediate domain,
and the essential component in order to close the structure-property loop, is polymer
characterization: what did we make, exactly?
There is no doubt that polymer characterization is, in general, a formidable experimental
challenge. At the root of the problem is the unavoidable fact that all synthetic polymers are het-
erogeneous in their molecular structure. A simple calculation reveals that even a tank car full of
homopolymer will not contain two molecules that are precisely identical. Not only is there a
distribution of molar mass, but there is also heterogeneity in multiple variables, including,
for example, regioisomerism, stereochemistry, long- and short-chain branching, and copoly-
mer sequence and composition. Ideally, we would like to characterize all of these distributions,
at least to the level of a mean value and a variance. This goal is currently beyond the capabilities
of most laboratories, although experimental science continues to advance significantly.
It is no surprise that full molecular characterization demands a suite of experimental tools.
Each technique will provide valuable information, but none can be sensitive to all of the vari-
ables of interest. This creates a further challenge for the polymer scientist: how to decide which
techniques to use, and how to use them effectively? It is tempting to view each instrument as a
black box: insert some sample material, and out pops an answer (typically with a ridiculous
number of significant figures). As experimental scientists, we know this is dangerous; every
technique relies on a set of assumptions, which may or may not apply for the sample we care
about. We can consult a textbook, or, more likely, Wikipedia, and find a brief discussion of the
technique, and a statement of the working equations. While helpful, this is not sufficient.
A good practitioner needs to understand the assumptions, and also to be conversant with
the strengths and weaknesses, the opportunities and blind spots, of each characterization tool.
This is the void that Molecular Characterization of Polymers: A Fundamental Guide aims to fill.
The authors adopt a tutorial style, so that specific prior knowledge is not required. And, by
taking this approach, the reader is empowered to acquire a deeper understanding of the
theory underlying each approach. As noted before, there have been substantial recent ad-
vances in instrumental techniques, and thus this up-to-date treatment will be very valuable
for experienced practitioners as well.
Timothy P. Lodge
University of Minnesota, Minneapolis, MN, United States

xi
 Preface
It is very appropriate as we celebrate in
2020 the 100-year anniversary of Nobel Lau-
reate Hermann Staudinger’s “discovery” of
polymers, that we assemble this new book
on Molecular Characterization of Polymers: A
Fundamental Guide. This is because Stau-
dinger, beginning in 1920 until 1930, utilized
a series of ingenious molecular characterization
studies to prove the long chain nature of poly-
mers, their high molecular weights, and their
distribution of chain lengths (molecular
weight distribution, MWD) [1–3]. This work
earned Staudinger the Nobel Prize in Chem-
istry in 1953.
In the decades after Staudinger’s discov-
ery of polymers, the synthesis of polymers
of ever-increasing complexity led to the
“polymer age” of materials which continues
to this day, and new methods have been
continually developed in order to better
characterize polymers. However, even in
1975, the eminent polymer scientist Fred
Billmeyer stated “…characterization of poly-
mers is inherently more difficult than that of
other materials. Polymers are roughly equiv-
alent in complexity to, if not more complex
than, other materials, at every physical level
of organization from microscopic to macro-
scopic…” and “We would wish, ideally, to
characterize all aspects of polymer structure
in enough detail to predict its performance
from first principles. I seriously doubt that
this will ever be possible…” [4].
Billmeyer’s remarks stem from the fact
that synthetic polymers always exhibit a
distribution of chain lengths or MWD.
Homopolymers are further complicated by
xiii
tacticity, mode of monomer insertion (e.g.,
head-to-tail or head-to-head), chain con-
formation (flexibility), end groups, and
branching (long chain and/or short chain).
Copolymers are further complicated by
monomer sequence distributions and varia-
tions in comonomer content across the
MWD. All of these factors affect polymer
processing and the properties of the poly-
meric material, but polymer characterization
methods only yield average values and dis-
tributions of key molecular parameters. Even
today, nearly a half century since Billmeyer’s
statements, despite all the advances in char-
acterization techniques and computation, the
thorough and accurate molecular characteri-
zation of polymeric materials remains highly
challenging.
Another factor complicating the thorough
characterization of polymers is the need to
employ a combination of different character-
ization techniques, as well as some knowl-
edge on how the polymer was synthesized,
in order to rigorously characterize even the
simplest of polymers. Unfortunately, scien-
tists actively involved in characterizing poly-
mers, either in industry or in academia, are
usually specialists in one or two analytical
techniques and lack detailed knowledge in
a wide range of complementary polymer
characterization techniques, as well as poly-
mer synthesis methods and their impact on
polymer structure, that must be employed
in order to understand the polymer’s molec-
ular structure in detail. As examples, in
industry analytical chemists are often
confronted for the first time with
xiv
characterizing polymers and they may have
little or no experience in characterizing such
complex mixtures. Also, few polymer scien-
tists are rigorously trained in a wide range of
polymer characterization techniques.
Thus the primary purpose of this book is
to serve as a textbook for a course (academic
course or short course) on polymer charac-
terization in order to better train the next
generation of polymer characterization
experts. This book is thus written in a tutorial
style to serve as an introduction to the vari-
ous polymer characterization techniques.
We anticipate that this book, written in this
style, will also be useful to scientists in indus-
trial polymer analysis laboratories who
are applying a characterization technique
to polymers for the first time. In addition to
fundamentals, we have also included in each
chapter recent advances in the technique,
information on instrumentation, and recent
applications to make this book useful to sci-
entists with experience in a technique but
looking for updates on recent advances and
applications.
This book begins with several chapters on
chromatography of polymers. Chapter 1 in-
troduces basic principles of chromatography
of polymer, including size exclusion chroma-
tography (SEC), high performance liquid
chromatography (HPLC), and liquid chro-
matography at the critical condition. Data
reduction methods and column technologies
are discussed. Chapter 2 discusses
multidetector SEC of polymers, using detec-
tors such as light scattering and viscosity de-
tectors, for characterizing simple and
complex (copolymer, branched) polymers.
SEC remains the workhorse for characteriz-
ing polymer molecular weight distributions.
Chapter 3 discusses the use of temperature
gradient interaction chromatography for
characterization of branched polymers
and copolymers, end functionalized poly-
mers, and isotopically labeled polymers.
Preface
Chapter 4 describes basic principles and ap-
plications of field flow fractionation (FFF) to
polymers. Chapter 5 focuses on the industri-
ally important area of characterization of
polyolefins, which constitute half of the an-
nual polymer production worldwide. Be-
cause of their limited solubility, polyolefins
present special challenges in their character-
ization. Multidetector SEC of polyolefins is
discussed, along with crystallinity-based
techniques such as temperature rising elu-
tion fractionation and crystallization analysis
fractionation.
Chapter 6 describes the use of combina-
tions of fundamental hydrodynamic ap-
proaches (analytical ultracentrifugation,
intrinsic viscosity, translational diffusion,
and SEC) to characterize molecular weights,
dimensions, and conformation. These com-
bined techniques are especially useful with
complex polymers such as polyelectrolytes.
Chapter 7 describes the use of viscometry
to measure polymer size, molecular weight,
as well as gather insight into conformational
characteristics and branching. Methods for
detecting and quantifying long chain
branching, including viscometry, light scat-
tering, and multidetector SEC are described
in Chapter 8.
Chapter 9 is focused on recent advances in
mass spectrometry of polymers, focusing on
MALDI-TOF-MS and MS/MS. Chapters 10
and 11 describe the use of vibrational spec-
troscopy and NMR for structural characteri-
zation of polymers, including end groups,
composition, tacticity, etc. Chapters 12 and
13 describe the use of static and dynamic
light scattering to characterize polymer mo-
lecular weights, sizes, thermodynamic inter-
actions and conformations. Chapter 14
introduces LenS3, a new light scattering de-
tector that measures polymer molecular
weights and allows for radius of gyration
measurements in the sub-10-nm range. The
use of X-rays and neutrons for probing
 Preface
polymer structure and conformation, in bulk,
thin film, and in solution, is described in
Chapter 15 along with selected applications.
Chapter 16 covers microscopy of polymers,
with a basic introduction to SEM, TEM, and
AFM and recent applications to polymers.
We are grateful to all the authors who
made the timely assembly of this book possi-
ble even under the challenges imposed by
the Covid-19 pandemic.
Muhammad Imran Malika, Jimmy Maysb, and
Muhammad Raza Shaha
a
University of Karachi, Karachi, Pakistan
b
University of Tennessee, Knoxville, TN,
United States
xv
References
€
[1] H. Staudinger, Uber polymerisation, Ber. Deut.
Chem. Ges. 53 (1920) 1073–1085.
[2] H. Staudinger, Uber € die Konstitution der
Hochpolymeren, Ber. Deut. Chem. Ges. 61 (1928)
2427–2431.
[3] H. Staudinger, W. Heuer, Uber € hochpolymere
Verbindungen, 33. Mitteilung: Beziehungen
zwischen Viscosit€at und Molekulargewicht bei
Poly-styrolen, Ber. Deut. Chem. Ges. 63 (1930) 222–
234.
[4] F.W. Billmeyer, Trends in polymer characterization,
J. Polym. Sci. Symp. 55 (1975) 1–10.
 C H A P T E R

1
Basic principles of size exclusion and
liquid interaction chromatography
of polymers
Muhammad Imran Malika and Harald Paschb
a
H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological
Sciences (ICCBS), University of Karachi, Karachi, Pakistan bDepartment of Chemistry and
Polymer Science, University of Stellenbosch, Stellenbosch, South Africa

Polymers are inherently complex multicomponent materials having several simple and
distributed properties. Simple properties include total weight of the polymer, residual mono-
mer/oligomer, gel content, etc. Distributed properties are those in which different molecules
of the same sample have dissimilar values. The important distributed properties of polymers
include molar mass, chemical composition, sequence length, end group functionality, molec-
ular topology, etc. The performance properties of polymers are highly dependent upon these
distributed properties. The performance of polymers for any particular application can be im-
proved significantly by carefully monitoring, adjusting, and understanding their molecular
distributions. An important tool for the determination of distributed molecular properties of
polymers is separation science.
The size, chemical composition, sequence of repeat units, and architecture are some impor-
tant parameters that need to be considered when analyzing any polymer. The constitution,
configuration, and conformation of macromolecules are also critical for regulating any poten-
tial application. Polymers having similar molar masses and chemical compositions can have
completely different properties depending upon the sequence, constitution, configuration,
and conformation of their repeat units. Polymers having any distribution beyond only molar
mass are termed as complex polymers.
The concept of molecular heterogeneity can be utilized to describe the structural complex-
ity of synthetic polymers, see Fig. 1.1. Different types of heterogeneities of polymer chains
might superimpose each other and a given polymer sample may exhibit a molar mass dis-
tribution, a chemical composition distribution, individual block length distributions,

Molecular Characterization of Polymers 1 Copyright # 2021 Elsevier Inc. All rights reserved.
https://doi.org/10.1016/B978-0-12-819768-4.00007-5
2 1. Basic principles of size exclusion and liquid interaction chromatography of polymers

Chain Length End Group

Polymer Concentration

Block Length Architecture

Molar Mass
FIG. 1.1 Molecular heterogeneity of complex polymers. Reproduced from H. Pasch, Hyphenated techniques in liquid
chromatography of polymers, Adv. Polym. Sci. 150 (2000) 1–66, with permission from Springer Nature. Copyright 2000.

a functionality type distribution (end group distribution), and a molecular architecture

distribution. Important information required for the comprehensive characterization of
complex polymers are the molar mass distributions within each type of heterogeneity.
Synthetic polymers always have a dispersity with regard to molar mass that originates
from the presence of polymer chains having different lengths. Molar masses of polymers
may be expressed as different averages, e.g., number-average molar mass (Mn), weight-
average molar mass (Mw), etc. The broadness of the molar mass distribution can be expressed
by the molar mass dispersity (Ð) that is the ratio of weight-average and number-average mo-
lar masses (Mw/Mn). Size exclusion chromatography (SEC) is one of the most important
methods for the analysis of molar mass distributions of polymers [1].
Different functional groups at the polymer chain end or along the polymer chain introduce
another heterogeneity that is particularly important for oligomers and telechelic polymers.
The properties and reactivities of oligomers and telechelic polymers differ with regard to
the number and nature of the functional groups. Spectroscopic techniques can only provide
average numbers, whereas well-designed chromatographic separation methods can reveal
the functionality distribution as a function of other heterogeneities, e.g., the molar mass
distribution [2].
The involvement of more than one monomer in a polymerization reaction complicates the
situation and the resulting polymer may have a chemical composition distribution
superimposing the molar mass distribution. Spectroscopic techniques such as NMR and FTIR
can provide an average chemical composition of the sample. However, no information on the
distribution of chemical composition as a function of molar mass can be obtained. Moreover,
spectroscopic techniques can typically not differentiate between copolymers and mixtures of
their respective homopolymers. Interaction chromatographic techniques can provide more
 1.1 Liquid chromatography of polymers 3
insight into the complex composition of copolymers. There can be several additional hetero-
geneities in copolymers such as the bulk molar mass distribution, the distribution of repeat
units in the copolymer, the lengths of different segments in case of segmented copolymers, the
presence of any type of homopolymer in the bulk sample, etc. The above-mentioned factors
make the comprehensive characterization of polymers a multifaceted task. For the analysis of
the chemical composition as a function of molar mass, multidetector approaches may be re-
quired (see Chapter 2 for detailed discussion). The independent analysis of each type of het-
erogeneity requires independent separation techniques that are selective regarding specific
types of distributions. In order to get access to one type of heterogeneity as a function of other
heterogeneities, coupling of independent chromatographic techniques or hyphenation of
chromatographic separation with spectroscopic techniques is imperative.
To summarize, a minimum of two independent methods are required for the analysis of
complex polymers each with a certain selectivity for one type of heterogeneity. In this context,
different modes of HPLC of polymers such as SEC, liquid chromatography at critical condi-
tions (LCCC), and interaction chromatography (IC) or liquid adsorption chromatography
(LAC) can be coupled to each other. Coupling of a chromatographic separation with spectro-
scopic techniques can also be a fascinating approach in order to obtain the distribution of one
property as a function of another distribution.

1.1 Liquid chromatography of polymers

The basic principle of any chromatographic process is based on the selective distribution of
the analyte molecules between the mobile and the stationary phase. The separation process
of chromatography can be described by
Ve ¼ Vi + Vp Kd (1.1)
where Ve, Vi, Vp, and Kd represent the elution (retention) volume of the analyte, the interstitial
volume of the column, the pore volume of the packing, and the distribution coefficient, re-
spectively. The distribution coefficient is the ratio of the analyte concentration in the mobile
and the stationary phase. Kd is related to the variations in Gibbs free energy Δ G that depends
on analyte partitioning between interstitial and pore volume [3].
ΔG ¼ ΔH  TΔS ¼ RT ln Kd (1.2)
The logarithmic plot of the distribution coefficient allows the determination of the entropic
(ΔS) and enthalpic (ΔH) contributions (van t’ Hoff plot):
ΔS ΔH
ln Kd ¼  (1.3)
R RT
Different effects that contribute to the change in Gibbs free energy are (1) the decrease
in conformational entropy that originates from limited dimensions inside the pores of the
stationary phase, and (2) changes in enthalpy that originate from the (adsorptive) interaction
of macromolecules with the stationary phase.
SEC separates macromolecules with regard to their hydrodynamic volume in dilute solu-
tion. The stationary phase in SEC is a swollen gel having a characteristic pore size
4 1. Basic principles of size exclusion and liquid interaction chromatography of polymers

distribution. The macromolecules may have less or more access to the pores depending on
their hydrodynamic sizes. Very large molecules cannot enter the pores and are excluded, elut-
ing at the interstitial volume Vi. Very small molecules have full access to the pores of the sta-
tionary phase and elute at the void volume of the column which is the sum of interstitial and
pore volume (Vo ¼ Vi + Vp). Hence, the separation range of SEC is 0 < KSEC < 1.
In ideal SEC, the distribution coefficient depends only on entropy changes without any
involvement of enthalpic interactions; however, in real SEC this is difficult to achieve. On
the other hand, the distribution coefficient in the case of IC totally depends on the interaction
strength of the analyte molecules with the stationary phase. This is perfectly true only for
small molecules. Longer chains of polymers may not have access to the whole pore volume,
hence, entropic factors must be assumed to contribute in addition to enthalpic contributions.
In case of polymers, often mixed modes of chromatography are operative and methods are
defined by the predominance of entropic or enthalpic interactions. Entropic interactions are
predominant in case of the size exclusion mode, i.e. T Δ S > Δ H corresponds to negative value
of Δ G, while separation in the interaction mode is dominated by enthalpic interactions, i.e.
Δ H > T Δ S corresponds to positive value of Δ G. Interaction forces exactly compensate
entropy losses at the transition point of the exclusion and interaction modes, i.e. Δ H ¼ T Δ S
corresponds to zero value of Δ G. This mode of liquid chromatography of polymers is often
termed as liquid chromatography at critical conditions.
Hence, Gibbs free energy at the chromatographic critical point is constant (Δ G ¼ 0) and the
value of the distribution coefficient equals 1, Kd ¼ 1, independent of the molar mass of the
polymer and the pore size of the stationary phase. A narrow range between size exclusion
and interaction modes of LC that is sensitive to changes in temperature and mobile phase
composition is related to the chromatographic critical point. This transition from one mode
of separation to the other was reported for the first time by Belenkii et al. [4] and Tennikov
et al. [5]. They demonstrated sudden changes in the elution behavior by slight variations in
the composition of the mobile phase. Hence, the transition point between the SEC and IC
modes can be realized by carefully adjusting the mobile phase composition and the temper-
ature. This specific transition point is labeled as the chromatographic critical point (CCP) and
the corresponding mode of liquid chromatography is termed as liquid chromatography at
critical conditions (LCCC).
A presentation of the transition between the three modes of liquid chromatography of
polymers is shown in Fig. 1.2. In SEC, retention decreases with increasing molar mass
whereas retention increases with molar mass in IC or LAC. At LCCC, the exclusion and in-
teraction effects are compensated rendering a molar mass independent elution of a particular
polymer at a constant elution volume. These separation modes can be combined in various
ways to realize separations of polymers with regard to different distributions such as molar
mass, chemical composition, and functionality. SEC, the most frequently used method for
polymer analysis, separates polymers with regard to their hydrodynamic size in dilute solu-
tion, and several approaches are in place to obtain chemical composition information as a
function of molar mass that include multiple concentration detector systems, and universal
calibration with viscometric and light scattering detection (see Chapter 2 for detailed discus-
sion). One must keep in mind, however, that SEC separation is based on size and the chemical
compositions obtained by different approaches are only average values related to a given SEC
fraction.
 1.2 Theory of polymer chromatography 5

FIG. 1.2 Dependence of elution volume on molar mass in different modes of liquid chromatography of polymers.

1.2 Theory of polymer chromatography

Retention of an analyte on the stationary phase in HPLC is expressed in terms of the

distribution coefficient as follows:
Ve  Vi
K¼ (1.4)
Vp
The distribution coefficient may assume different values in different modes of liquid
chromatography. In this context, de Gennes introduced a function that is termed the interac-
tion parameter, c [6, 7]. The value of interaction parameter depends on the mobile phase
composition (for a given polymer-stationary phase system) and temperature. The unit of
the interaction parameter is inverse length (nm1).

1.2.1 Size exclusion chromatography

In SEC, the value of the interaction parameter c is negative while the distribution coefficient
assumes values between zero and one (0 < K < 1). SEC separation is controlled by entropy
changes induced by differently sized species moving through the chromatographic column.
The elution volume of SEC in ideal conditions is given by Eq. (1.5) [8]
 
4 R
VSEC ¼ Vi + Vp 1  pffiffiffi (1.5)
πD
6 1. Basic principles of size exclusion and liquid interaction chromatography of polymers

where R is the radius of gyration of the analyte while D is the pore diameter of the stationary
phase. The radius of gyration expressed in terms of length and number of repeat units is
given as
rffiffiffi
n
R¼a (1.6)
6
where a is the length and n is the number of the repeat units.
Separation in SEC is realized with regard to molecular dimensions regardless of compo-
sition and functionality.

1.2.2 Interaction chromatography or liquid adsorption chromatography

IC is based on the strength of interaction of the analyte molecules with the stationary phase.
The value of c is positive whereas the distribution coefficient has values larger than one (k > 1)
that increases exponentially with the number of repeat units.
Retention in IC is often described in terms of Martin’s rule [9, 10].
ln k ¼ A + Bn (1.7)
The dimensionless factor k is deduced from the elution volume of the solute and holdup
volume (different from void volume) of the column. An excellent review with regard to
holdup volume of the column is presented by Rimmer et al. [11]. In this context, Gorbunov
and Skvortsov developed a theory of chromatography for flexible homopolymers that can
interact with the stationary phase [12, 13]. According to this theory, the elution volume of
a nonfunctional polymer chain in IC is given by
 
4 R 2Vp 2Vp
Ve ¼ Vi + Vp 1  pffiffiffi  + exp ðcRÞ2 ½1 + erf ðcRÞ (1.8)
πD cD cD
wherein D is the pore diameter of the stationary phase, R is the radius of gyration of the mac-
romolecule, c is the interaction parameter, and erf(cR) is the error function. It is pertinent to
mention here that the value of c is independent of D, Vp, Vi, or R (i.e., the degree of polymer-
ization n).
The term erf(cR) approaches unity at sufficiently strong interaction (typical for IC) and,
therefore, Eq. (1.8) can be rewritten as
 
4 R 2Vp 2Vp
Ve ¼ Vi + Vp 1  pffiffiffi  + exp ðcRÞ2 (1.9)
πD cD cD
or
 2 2
4Vp c a
Ve ¼ Vo∗ + exp n (1.10)
cD 6
where V∗o is the accessible volume for the polymer chain.
 
4R 2Vp 2Vp
Vo∗ ¼ Vi + Vp 1  pffiffiffi  ¼ VSEC  (1.11)
D π cD cD
 1.2 Theory of polymer chromatography 7
Obviously, the accessible volume is smaller than the void volume (Vo ¼ Vi + Vp). The acces-
sible volume in IC can be obtained by plotting the elution volumes of oligomers Vn with n
repeat units versus the difference in elution volume of consecutive peaks (Δ Vn ¼ Vn  Vn1)
Vn ¼ Vo∗ + γΔVn (1.12)
The accessible volume is represented by the intercept of the plot, and slope γ ¼ e /(eB  1)
B

can be used to calculate the interaction parameter c, wherein B ¼ (c2a2)/6 is the slope in
Martin’s rule (see Eq. 1.7)
rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
1 γ
c¼ 6 ln (1.13)
a γ1
It can be noticed that the number n of the individual peaks is not required for the
determination of the accessible volume and the interaction parameter, provided sufficiently
strong interaction is present. Moreover, Gorbunov et al. developed a software for the deter-
mination of interaction parameters in all modes of LC [14]. The approach is based on a set of
measurements of nonfunctional polymer standards of known molar mass. Eq. (1.10) can also
be rewritten in terms of the retention factor k∗
 2 2
Ve  Vo∗ 4Vp c a
k∗ ∗
¼ ∗
exp n (1.14)
Vo cDV 0 6
The logarithmic form of the equation corresponds to Martin’s rule.
 
4Vp c 2 a2
lnk ¼ ln
∗ + n (1.15)
cDV ∗0 6
Martin’s rule can also be rewritten for nonfunctional chains as
2Sp ðcaÞ2
ln k∗ ¼ ln + n (1.16)
cV ∗0 6
The pore surface can also be determined from the intercept of Martin’s plot once the
interaction parameter is determined using the earlier equations. However, the identification
of the peaks is required for the determination of the pore surface. For mono-functional chains,
an additional parameter q is required [15]. The specific end group parameter q measures the
difference of free energy of adsorption of end group and repeat unit [16]. A facile method
for the determination of q has been elaborated by Nguyen and Trathnigg [17].
In IC, retention of mono-functional chains with an adsorbing end group can be written as
!
4 Vp c 2 a2
ln k∗m ¼ ln ∗
ð1 + qcÞ + n (1.17)
cD V0, m 6

V∗0,m is the accessible volume for mono-functional chains that is smaller than the accessible
volume for nonfunctional chains.

∗ ¼ V∗ 
Vp 2 q
V0,m pffiffiffi (1.18)
0
D π Rc
8 1. Basic principles of size exclusion and liquid interaction chromatography of polymers

The value of k increases exponentially with the number of repeat units. Straight lines
with the same slopes are obtained in a plot of lnk vs n in both cases, having rather different
intercepts.
IC allows for separation of oligomers of nonfunctional polymers as well as mono-
functional polymers with a rather weakly adsorbing end group. Stronger interaction of the
end group results in poor resolution of individual oligomers.

1.2.3 Liquid chromatography at critical conditions

Entropic and enthalpic terms compensate each other in liquid chromatography at critical
conditions. At this so-called chromatographic critical point (CCP), the values of the distribu-
tion coefficient and the interaction parameter equal to one and zero, respectively. The com-
pensation of enthalpic interaction and entropic exclusion renders molar mass-independent
elution of nonfunctional chains at the void volume of the column. An additional term qa is
required to describe the influence of an interacting end group a on retention. The elution vol-
ume of chains with an adsorbing end group at their chromatographic critical point is larger
than the void volume, however, independent of molar mass [16, 18].
Va  Vi + Vp ð1  qa Þ  V0 + qa Vp (1.19)
The contribution of the end group on retention at the critical point allows for separation of
mono-functional chains with regard to the interaction strength of the end group independent
of molar mass. However, a very different behavior is observed with di-functional chains (with
adsorbing groups at both ends) [16, 19]. The elution volume of chains with symmetrical
groups at both ends is given by
 
q2a D
Vaa  Vi + Vp 1 + 2qa + pffiffiffi (1.20)
πR
The equation indicates that di-functional chains are separated with regard to the radius of
gyration R of the critical polymer chain. Asymmetrical di-functional polymer chains (bearing
different groups at the chain ends) hold the same relation,
 
qa qb D
Vab  Vi + Vp 1 + qa + qb + pffiffiffi (1.21)
πR
where a and b are end groups.
Di-functional chains elute later than mono-functional polymeric chains; however, their
elution volume is not only dependent on contributions of the end groups a and b but also
contain an additional term that is the ratio of the pore diameter D of the stationary phase
and the radius of gyration R of the polymer molecules [16, 19]. Consequently, elution of
di-functional polymer chains with adsorbing end groups follows SEC order.
There is another special situation where the end group adsorbs while the repeat units are in
SEC mode. This regime of liquid chromatography of polymers is termed as liquid exclusion
adsorption chromatography (LEAC) [20]. Under these conditions, the interaction parameter
of the repeat unit c is negative while the interaction parameter of the end group cB is positive.
This condition renders elution of mono-functional chains in SEC order beyond the void
 1.3 Thermodynamics of polymer chromatography 9
volume of the column. The mathematical relation for the elution volume VAB of short
mono-functional polymer chains under the described conditions is given by
 pffiffiffi 
π pffiffiffiffiffiffi
VAB  VB 1  cB RA ¼ VB ð1  C nA Þ (1.22)
2
where A is the repeat unit while B is the end group.
The elution volume decreases with increase in the number of repeat units (and hence
radius of gyration). The method can be used for oligomer separations of mono-functional
polymer chains containing 10–15 repeat units in SEC order beyond the void volume of the
column. This is essentially an isocratic separation that allows a RI detector to be employed,
resulting in accurate quantification [21].

1.3 Thermodynamics of polymer chromatography

The distribution coefficient depends on changes in Gibbs free energy that correspond to
variations in entropy and enthalpy, see Eqs. (1.2) and (1.3). Separation in the size exclusion re-
gime is governed by the entropic term whereas interaction is an enthalpic process. However,
entropic or enthalpic contributions are not easy to avoid completely especially in the case
of macromolecules. Both enthalpic and entropic terms compensate each other at the critical
mode of liquid chromatography of polymers which means Δ G ¼ 0, as Δ H ¼ T Δ S.
As described previously, SEC and IC mechanisms may show different dependences on
temperature. The distribution coefficient solely depends on entropic changes in ideal SEC
(no enthalpic interactions) rendering no dependence on temperature. In LCCC, entropic
and enthalpic effects are counterbalanced, hence, any change in temperature would require
a different mobile phase composition to retain the critical behavior. Consequently, retention
in IC depends both on enthalpy and entropy changes. Retention of any polymer on a given
stationary phase depends on the mobile phase composition and the temperature. However,
the extent and direction of this dependence varies.
The changes in entropy and enthalpy can be determined from the van’t Hoff plot, ln K vs
1/T. Various approaches that primarily differ in the calculation of the distribution coefficient
are used for the determination of thermodynamic parameters [22, 23].
Direct proportionality between the distribution coefficient K and the retention factor
k ¼ (Ve  V0)/V0 is often applied in this regard
ΔH° ΔS°
lnk ¼  + + lnφ (1.23)
RT R
wherein term lnφ corresponds to mobile and stationary phase ratio (generally not known),
principally indicating the pore volume and interstitial volume. Hence, the slope and
intercept in a plot of ln K vs 1/T represent the thermodynamic parameters Δ H°/R and
(Δ S∗/R) ¼ (Δ S°/R) +lnφ. However, there is no direct correlation between the distribution
coefficient K and the retention factor k as is clear from equations
Ve  V0
k¼ (1.24)
V0
10 1. Basic principles of size exclusion and liquid interaction chromatography of polymers

Ve  Vi Ve  Vi
K¼ ¼ (1.25)
Vp V0  Vi
The relationship between the distribution coefficient K and the retention factor k can be
devised as
Vp
k ¼ ð K  1Þ (1.26)
V0
Contrary to a typical assumption, there is no direct proportionality between K and k. The
assumption that K ¼ kφ is an approximation and holds only for K ≫ 1. The exact relation con-
tains the distribution coefficient K; however, the determination of characteristic volumes Vi,
Vp, and V0 are required.
It is pertinent to mention here that the determination of the value of void volume is not
trivial. Numerous articles address the issue of the accurate determination of the void volume,
the dead volume, and the holdup volume [11, 24], having different definitions relevant to par-
ticular situations [11, 25–27]. The void volume is usually taken as the total amount of solvent
in the column that can be determined gravimetrically. On the other hand, the holdup volume
is considered as the elution volume of an unretained compound that can be determined by
various methods [11, 24]. The interstitial volume can be determined by inverse SEC. It is ac-
tually the elution volume of a completely excluded polymer from the pores of the stationary
phase. However, these values are very much dependent on the mobile phase and may assume
dissimilar values in different mobile phases.

1.4 Equipment and materials

Separations by different modes of liquid chromatography of polymers pose several chal-

lenges that can be addressed by state-of-the-art HPLC instrumentation. Typically, a flexible
pump (for isocratic and gradient separations), a column oven (for stable temperature
conditions), and a reliable set of detectors (for quantitative information with regard to differ-
ent molecular parameters) are required.
The main components of any HPLC instrument include a solvent delivery system, a sam-
ple injector, a set of detectors, and a data acquisition system. Columns are the core of any
separation system that contain stationary phases of different nature, which are selected
according to the targeted separation.

1.4.1 Solvent delivery and injector

One of the most important requirements of any HPLC system is a constant and reproduc-
ible supply of mobile phase. Several types of pumps are employed for a reliable mobile phase
delivery in HPLC. These include syringe pumps (works like a syringe for pulseless flow) and
reciprocating pumps that exist in various modifications as single pistons, parallel dual pis-
tons, and dual pistons in series. General requirements of HPLC/SEC with regard to the pump
include a flow rate precision of 0.2%, a pressure output of 6000 psi, a pressure pulsation of less
 1.4 Equipment and materials 11
than 1% at 1 mL/min, a flow rate range of 0.01–10 mL/min, chemical resistance to a wide
range of solvents, and small holdup volumes for rapid solvent exchange. Mobile phases used
for HPLC are generally degassed by inline degassers to get rid of dissolved gases. An alter-
native is the degassing of the mobile phase by ultrasonication prior to use.
The selection of the pump for any HPLC system depends on the intended application. Sy-
ringe pumps are suitable for SEC and LCCC due to their flow stability and minimum evap-
oration of the solvent. However, syringe pumps are not suitable where high pressure mixing
is required. Reciprocating pumps allow low pressure mixing and are the preferred choice for
gradient elution. Recently, reciprocating pumps in different variations are almost exclusively
employed for HPLC by all the manufacturers.
The injection of sample as a narrow band is imperative to avoid peak broadening from in-
jection. This requires a sharp plug of sample solution injected into the mobile phase. Sample
injection is typically conducted through a two-position, six-port valve which may be operated
manually or automatically. For precise measurements, the sample loop should be completely
filled. The size of the sample loop depends on the column dimensions, the sensitivity of the
detectors, and the nature of separation. Larger loops (50–100 μL) with dilute sample solutions
are recommended for SEC rather than using higher concentrations with smaller loops. On the
other hand, higher concentrations are preferred using smaller loops (10–50 μL) for IC and
LCCC. Modern instruments are equipped with autosampling devices that allow analysis
of multiple samples without intervention of operator. Most commercial autosamplers permit
injection of any volume in the range 0–2000 μL with a precision of 0.5%. Additionally, mod-
ern autosamplers are also equipped with sample filtration, variable speed and temperature
mixing, needle wash, etc.

1.4.2 Column dimensions

Types and dimensions of chromatographic columns depend on the mode of operation. In
contrast to other modes of HPLC, separation in SEC is predominantly determined by the type
of stationary phase with minimal influence of the mobile phase. The separation takes place in
the pore volume that equals 30%–40% of total elution volume. Therefore, for achieving fairly
good separation efficiency, longer columns (25–60 cm) with larger volumes of stationary
phase are required. Typically, a multiple-column set is used for SEC analysis. Inner diameters
of commercially available columns are in the range of 5–8 mm for analytical SEC and
22–25 mm for semipreparative SEC. The plate height decreases with a decrease in particle size
resulting in a higher number of plates per unit length. SEC columns are classified into
microbore, narrow bore, analytical, semipreparative and preparative with increase in particle
size, column diameter, and length in the same order. Separation efficiency of any chromato-
graphic system is expressed in terms of number of theoretical plates, where a theoretical plate
refers to a single equilibrium step. Higher numbers of theoretical plates refer to better sepa-
ration. In this context, plate height, H, is obtained by dividing the length of column by the
number of theoretical plates. Smaller particles are preferred to allow higher packing densities
hence providing higher plate numbers; however, this results in an increased column back
pressure. The back pressure should not exceed 150 bar for most of the packings. Besides
the packing density of the stationary phase the column back pressure also depends on the
12 1. Basic principles of size exclusion and liquid interaction chromatography of polymers

viscosity of the mobile phase. As a rule of thumb, 1 mL/min should be the flow rate of a col-
umn with 8 mm inner diameter while 0.25 mL/min for a column with 4 mm inner diameter.
The total column length should be adjusted according to the back pressure produced in a
given mobile phase at optimum flow rate. Higher column temperatures are preferred as a tool
to reduce the back pressure compared to lower flow rates due to the fact that lower flow rates
result in a rapid increase in plate height and thus decrease in separation efficiency.
On the other hand, using smaller (high efficiency) columns is a trend in HPLC compared to
SEC where high efficiency can only be achieved using longer columns. Retention in HPLC
depends on the distribution coefficient of the analyte between the mobile and the stationary
phase. In contrast to SEC, separation in HPLC is governed by the composition of the mobile
phase. Smaller columns lead to faster analysis and lower solvent consumption. There are,
however, limitations of miniaturization due to the fact that smaller particles giving higher
separation efficiency result in an increased back pressure. Furthermore, the lengths and di-
ameters of connecting capillaries and the internal volume of the detector cell have to be small
for an acceptable overall efficiency of the system. Small sized particles result in smaller plate
height that in turn increases the number of theoretical plates per unit volume. Microbore
HPLC requires specialized injection systems, capillaries, and detectors. Narrow bore columns
can be operated with normal HPLC systems.

1.4.3 Stationary phases

Stationary phases are selected with respect to the intended separation. Porous silica or
cross-linked organic gels are commonly used stationary phases for SEC. Styrene-
divinylbenzene copolymer is the most widely employed stationary phase for SEC in organic
solvents. Modified silica or cross-linked hydrophilic polymers are a common choice for aque-
ous SEC. Nonetheless, polymer packings are available that can be used for a large variety of
different mobile phases. Generally, silica-based packings are rather robust compared to or-
ganic polymer-based packings. Commercially available column packings for organic and
aqueous SEC having varieties of pore sizes and applicable molar mass range are produced
by several companies such as Agilent, Macherey-Nagel, Tosoh Haas, Polymer Laboratories,
Merck, Phenomenex, Polymer Standards Service, Shodex, Malvern, and Waters. Commer-
cially available columns can be subdivided into two major categories, namely single pore col-
umns and mixed columns (also termed as linear). Fig. 1.3A demonstrates the elution volume
of narrow molar mass PS standards as a function of molar mass on SDV columns of various
pore sizes. As can be noticed, the calibration curve on single pore sized columns is not linear.
Approximately two decades of molar masses (e.g., 103–105 g/mol) are covered by traditional
single pore size columns. Recently, the use of mixed bed columns, made of mixing particles of
different pore sizes, is in fashion. Linear or mixed columns allow to cover a wider molar mass
range and the obtained plot of elution volume as a function of molar mass is linear, Fig. 1.3B.
Traditional HPLC columns that are used for the separation of low molar mass organic com-
pounds are equally applicable to interactive modes of liquid chromatography of polymers. As
a general rule, spherical particles are superior to irregular particles. The efficiency of a column
increases with decreasing particle size of the stationary phase that, on the other hand, leads to
an increased back pressure. SEC is solely based on the limited accessibility of the polymer
 1.4 Equipment and materials 13

107 A
107
106 A
105 A
106
104 A
Molar Mass (Da)
103 A
105 500 A
100 A
50 A
104

103

102
5 6 7 8 9 10 11 12
(A)
Elution Volume (mL)

107

106 Linear XL
Linear M
Linear S
Molar Mass (Da)

105

104

103

102
5 6 7 8 9 10 11
(B) Elution Volume (mL)
FIG. 1.3 Elution volume as a function of molar mass of PS standards on SDV columns in THF (A) single pore size
columns, (B) linear or mixed columns. Data provided by Polymer Standards Service [28].

molecules to the pores of the stationary phase, while separation in IC is based on the available
surface area of the stationary phase. In LCCC, both adsorption/interaction and exclusion
effects are compensated, hence pore size is very important.
The nature of the required stationary phase is basically determined by the separation
problem. Stationary phases for HPLC are mostly based on silica or cross-linked polymers.
14 1. Basic principles of size exclusion and liquid interaction chromatography of polymers

A general classification is based on the polarity of stationary and mobile phases. The station-
ary phase is polar compared to the mobile phase in normal phase chromatography (NP). On
the contrary, the mobile phase is polar compared to the stationary phase in reversed phase
chromatography (RP). Typically, plain silica having silanol groups is modified with dimethyl
silane having a variety of R groups. The R group determines the nature of the stationary
phase. Some typical stationary phases for both normal and reversed phase chromatography
are shown in Fig. 1.4. Stationary phases based on modified silica are typically used in RP chro-
matography. These are obtained by reacting silica with silanes. The reaction of silica with
silanes is seldom complete resulting in residual silanol groups in the stationary phase. These
residual silanol groups may affect the separation especially in the analysis of basic com-
pounds such as amines. A current trend to overcome this problem is the application of pack-
ings with a high carbon load and a high degree of end-capping. Nonetheless, polymer-based
packings such as cross-linked styrene-divinylbenzene copolymers may be the better choice to
overcome this limitation. It is worth mentioning here that nitrile-modified phases can be used
either as normal or reversed phases depending on the polarity of the mobile phase.

1.4.4 Mobile phases

Single solvents are typically used as mobile phases for SEC. The mobile phase for SEC
should be a thermodynamically good solvent for the polymer to be analyzed. Additionally,
it should be able to solvate the stationary phase, should be chemically inert, have a low

FIG. 1.4 Typical polar (normal phase) and nonpolar (reversed phase) stationary phases for liquid
chromatography.
 1.4 Equipment and materials 15
viscosity, be UV transparent, and possess an appropriate refractive index and low toxicity.
The mobile phase is selected with respect to the nature of the polymer and the stationary
phase. Frequently employed organic solvents for SEC analysis are tetrahydrofuran, chloro-
form, toluene, esters, ketones, dimethylformamide, etc. Low molar mass electrolytes can
be added to minimize nonexclusion effects in the analysis of polar polymers such as polyelec-
trolytes. Polyolefins are not soluble in any solvent at room temperature and, hence, require
separation at high temperatures in high boiling solvents, trichlorobenzene being the most
widely used mobile phase for polyolefins (see Chapter 5 for detailed discussion).
The primary criterion for the selection of a mobile phase for interactive modes of HPLC
includes sample solubility and complex interactions between the sample, the stationary
phase, and the solvent. Solvents are classified with regard to their chemical nature and po-
larity. The most important criterion for the selection of a mobile phase in IC is polarity. Sol-
vents are classified in terms of “eluotropic series” with regard to polarity. Although varying
values for solvent polarity are reported by different sources, the order remains the same. It is
worth mentioning here that solvent polarity may vary within chemically similar classes of
solvents. Typical HPLC solvents with their polarity index and UV cutoff are listed in
Table 1.1. Another important concern when selecting a solvent for any separation system
is its mode of detection. The spectroscopic behavior of a mobile phase needs to be considered
when spectrometric detectors such as UV, FTIR, or NMR are used.

1.4.5 Detectors
The separation of the sample in the column has to be monitored by one or more detectors
whose signal must represent the concentration of the polymer. Typical HPLC detectors for
low molar mass analytes are equally applicable to SEC/HPLC of polymers. However, there
are specific requirements and approaches that are associated with the peculiar nature of large
molecules. Detectors used in HPLC of polymers can be broadly classified into two major cat-
egories, concentration sensitive detectors and molar mass detectors. Additionally, there are
spectroscopic detectors that can provide direct chemical composition information.

1.4.5.1 Concentration sensitive detectors

The concentration of the solute in the solvent is directly related to the detector signal in
concentration sensitive detectors. These detectors can be classified into two major groups,
namely, selective detectors that measure a property of a solute, and universal detectors that
measure a bulk property of the mobile phase. Combinations of detectors may be required for
the analysis of copolymers.

Selective detectors
Several selective detectors are available in HPLC; however, not all of them are applicable to
polymers. The UV detector is the most widely employed selective detector in polymer anal-
ysis. IR detectors can be useful but are limited to mobile phases that do not absorb radiation at
the detection wavelength. The introduction of an evaporative interface provides a good alter-
native for offline coupling to FTIR [29, 30]. The eluate is sprayed on a Germanium disk that is
rotated at a particular speed. The disk is then transferred to the FTIR spectrometer to yield
16 1. Basic principles of size exclusion and liquid interaction chromatography of polymers

TABLE 1.1 Typical solvents used in liquid chromatography of polymers.

Class Solvent Polarity index UV cutoff
Alkanes Hexane, heptane 0.0 200

Cyclohexane 0.2 200

Aromatics Benzene 2.7 280
Toluene 2.4 285
Xylene 2.5 290
Ether Diisopropyl ether 2.2 220
Methyl-tert-butyl ether 2.5 210

Tetrahydrofuran 4.0 215

Dioxane 4.8 215
Alkyl halides Tetrachloromethane 1.6 263
Dichloromethane 2.5 235
Dichloroethane 4.0 225

Trichloromethane 4.8 245

Esters Butyl acetate 4.0 254
Ethyl acetate 4.4 260
Ketones Methyl ethyl ketone 4.7 329
Acetone 5.1 330
Alcohols n-Butanol 3.9 215

i-Propanol 3.9 210

n-Propanol 4.0 210
Methanol 5.1 205
Nitriles Acetonitrile 5.8 190
Amides Dimethylformamide 6.4 268
Carboxylic acids Acetic acid 6.2 230

Water 9.0 200

Reproduced from H. Pasch, B. Trathnigg, Multidimensional HPLC of Polymers, Springer, Berlin-Heidelberg-New York,
2013, with permission from Springer Nature. Copyright 2013.

spectral information as a function of elution time of the chromatogram. This setup must,
however, be combined with an additional concentration sensitive detector for accurate quan-
tification. Fluorescence and electrochemical detectors are other selective detectors for HPLC
that are not applicable to polymers.
The UV detector is the most familiar solute property detector. It is commercially available
in different modifications. The UV detector is based on the principle of absorption of light of a
 1.4 Equipment and materials 17
selected wavelength by the chromophore-containing analyte. The typical wavelength range
in this context is 180–350 nm. UV detectors can only be applied to solvents with low UV cutoff.
There are basically three types of UV detectors, namely, fixed wavelength detectors, variable
wavelength detectors, and diode-array detectors (DAD). Fixed wavelength detectors are
mostly equipped with a lamp emitting light at 254 nm. Variable wavelength detectors allow
for selection of a particular wavelength by means of a holographic grating. DAD allows for
simultaneous measurement of the whole UV spectrum over the entire chromatogram.

Universal detectors
Universal detectors measure the change in a bulk property of the mobile phase. Important
universal detectors are refractive index, conductivity, density, and evaporative detectors.
Universal detectors are less sensitive compared to selective detectors; however, they are com-
monly applied in analysis of polymers. The most widely employed detector in this regard is
the RI detector that is available in many modifications. Applications of the conductivity de-
tector to polymers are not common. Density detector works on the mechanical oscillator prin-
ciple and is an effective detector in polymer analysis especially in combination with other
detectors. Evaporative detectors are based on the vaporization of the volatile component
of the eluate (typically the solvent) and the detection of the nonvolatile components by scat-
tering of the transversal light beam.

RI detector
Three types of RI detectors are available, namely deflection refractometers, Fresnel refrac-
tometers, and interferometric refractometers. Deflection refractometer is the most commonly
employed detector in this regard. Deflection refractometers have a large cell but a better linear
range compared to Fresnel refractometers. Sensitivity of interferometric refractometers is
higher by one order of magnitude compared to other RI detectors. The response factor of
RI detectors is dependent upon molar mass as well as on chemical composition. Hence, an
additional concentration detector is required for analysis of copolymers and polymer blends.
Moreover, preferential solvation of one component of copolymer may affect the detector
signal [1].

Density detector
Density detector in combination with UV or RI detectors reveals additional information
in isocratic elution mode. It works on the mechanical oscillator principle [31, 32]. A typical
density detector consists of an oscillating U-shaped capillary whose period depends upon
the density of the content. However, there are no recent applications of density detector
for analysis of polymers.

Evaporative light scattering detector

The ELSD can be regarded as universal detector since it detects any nonvolatile compo-
nents of the eluate [33, 34]. Only few companies offer ELSDs compared to a wide range of
available UV detectors. In such instrument, the eluate is nebulized and the solvent from
the droplet is evaporated. Particles are formed by the nonvolatile components of each droplet
that scatter the incident light beam in a photodiode cell.
18 1. Basic principles of size exclusion and liquid interaction chromatography of polymers

ELSD offers a number of advantages for IC and LCCC; however, there are still several ca-
veats regarding quantification. Firstly, the linearity of ELSD signal is rather poor. Further-
more, the dependence of its response on molar mass and chemical composition is not yet
fully understood. The conversion of the detector signal to concentration is more complex
for ELSD compared to other detectors. The number, size, and refractive index of the particles
affect the intensity of scattered light. It is also influenced by the molar mass and the chemical
composition of the sample. Moreover, the size of droplets formed in the nebulizer depends on
the operating conditions and chemical nature of the sample. Thus numerous external param-
eters influence the performance of ELSD such as flow rate of the carrier gas and eluate,
oven temperature, viscosity, and surface tension of the eluate. Consequently, ELSD must
be calibrated carefully for reliable results [35, 36].

1.4.5.2 Molar mass sensitive detectors

In SEC, molar mass sensitive detectors are often used since they yield molar mass infor-
mation of each slice of a SEC peak. Molar mass sensitive detectors have to be combined with
conventional concentration detectors since their response depends both on molar mass and
concentration of the analyte. These detectors are classified into viscometric and light scatter-
ing detectors [1, 37]. The variety of light scattering detectors include low-angle light scattering
detectors (LALLS), two-angle light scattering detectors (TALLS), right-angle light scattering
detectors (RALLS), and multiangle light scattering detectors (MALLS). However, modern
applications involving the utilization of light scattering detectors for polymer analysis are
almost exclusively based on MALLS. The synergistic combination of viscosity and light scat-
tering detectors reveals very important information that is not accessible by using them sep-
arately [38]. Absolute molar mass distributions can be obtained by light scattering detection.
Radii of gyration can also be obtained provided more than one angle instrument is used.
On the other hand, intrinsic viscosity distributions are obtained by SEC with viscosity detec-
tion. For highly reliable results, a combination of a concentration detector with both viscosity
and light scattering detectors are employed. This three detector combination is capable of
revealing information with regard to branching [39, 40]. Detailed information with regard
to construction and working principle of viscosity and light scattering detectors can be found
in Chapter 2.

1.5 Typical situations in liquid chromatography of complex polymers

For a copolymer system consisting of two repeat units A and B, the values of interaction
parameters cA and cB of the individual structural units (A and B) have different values.
The total molar mass distribution (MMD) of the copolymer can be determined by SEC if both
cA and cB are negative. In this case, retention solely depends on the hydrodynamic volume
that increases with the molar mass. The retention in SEC decreases with increase in molar
mass showing minor influence of chemical composition. However, similar to the interaction
parameters, the SEC calibration functions for different polymers may also be different.
Moreover, dual concentration detection can be employed for determination of chemical com-
position as a function of molar mass [32, 41–44]. SEC separation of copolymers is applicable
 1.5 Typical situations in liquid chromatography of complex polymers 19
to random, statistical and block copolymers. However, discussion regarding other combina-
tions of interaction parameters is applicable to block copolymers or functional polymers only.
If both cA and cB are positive, reasonable separation in LAC or IC can only be achieved if one of
the blocks is monodisperse [45–48]. On the other hand, several superimposed series of peaks
will be obtained if both blocks are polydisperse and cA 6¼ cB.
Another important situation may arise where the interaction parameter for one repeat
unit is positive while it is negative for the other. In this case, each polymer homologues series
elutes in SEC order, but with Ve ≫ Vo. This special mode of liquid chromatography of poly-
mers, termed as liquid exclusion-adsorption chromatography (LEAC), can be utilized in the
separation of mono-functional oligomers with monodisperse adsorbing blocks or end
groups [20, 49–52]. LEAC is only applicable as second dimension in the analysis of block
copolymers [53].
In the same context, there can be a situation when the value of interaction parameter of one
of the structural units (cA or cB) equals zero that renders the corresponding block “chromato-
graphically invisible” and allows for the separation that is majorly influenced by the other
segment. Chromatographic invisibility refers to the situation where molar mass discrimina-
tion of a specific type of homopolymer is suppressed and the whole range of molar masses of
that particular homopolymer elutes at a similar elution volume. This suppression of molar
mass selectivity of one block renders the separation of the block copolymer with regard to
the other so-called noncritical block. The separation of the “visible” block may follow a
SEC [32, 47, 54–56] or LAC/IC mechanism [47, 57–60] depending on the interaction parameter
of the “visible” repeat unit.
Block copolymers refer to polymers consisting of two or more chemically different poly-
meric segments that are covalently joined. These blocks have different physical properties
that allow the synergistic combination of required properties in one product. Furthermore,
the blocks can be completely different with regard to polarity and in turn their solubility
in different solvents, rendering to special properties such as self-assembly into micelles. Dif-
ferent polarities of polymeric blocks result in varying compatibilities with stationary phases
of different nature. Hence, if chromatographic selectivity as a function of molar mass of a par-
ticular polymer is nullified, the retention of a block copolymer will solely depend upon the
chromatographic behavior of the other block. This is the principle of liquid chromatography
at critical conditions referring to zero value of interaction parameter for the critical homopol-
ymer. This point of zero selectivity with regard to molar mass of a particular polymer is also
referred to as the chromatographic critical point (CCP). A schematic presentation of the dif-
ferent chromatographic situations in LCCC of diblock copolymers is demonstrated in Fig. 1.5.
As mentioned earlier, chromatographic invisibility of the repeat unit is realized when the in-
teraction parameter is zero. The noncritical block may have a positive or a negative value of
the interaction parameter that leads to elution of the block copolymer after or before the crit-
ical homopolymer, respectively. Different situations can be encountered depending on the
polarities of the polymeric blocks, mobile phase and stationary phase.
In a mobile phase of rather high polarity, the establishment of CCP of a polar polymeric
segment is possible on both reversed and normal phase columns. However, the nonpolar seg-
ment in such a mobile phase will have an opposite elution behavior at CCP of the polar seg-
ment on stationary phases of opposite nature. On a NP column at the CCP of the polar block in
a polar mobile phase, the nonpolar block will be excluded allowing elution of the block
20 1. Basic principles of size exclusion and liquid interaction chromatography of polymers

FIG. 1.5 Schematic representation of differ-

ent chromatographic situations in LCCC of
diblock copolymers related to the polarity of
polymeric blocks, mobile phase and
stationary phase.

copolymer in exclusion regime. On the other hand, in a polar mobile phase at CCP of the polar
block on a RP column the nonpolar block will have stronger interaction with the stationary
phase that will result in elution of the block copolymer in interaction regime. On the same
lines, CCP of nonpolar polymers is possible on both RP and NP columns in a mobile phase
of low polarity. Polar polymers at CCP of the nonpolar polymer will elute in exclusion regime
on a RP column while in interaction regime on a NP column, Fig. 1.5 [2, 56, 61]. As can be
imagined, when the polymeric blocks of different nature are connected to each other, they
cannot elute separately but only together. Nonetheless, the molar mass of the critical polymer
would not affect the elution behavior of the noncritical polymer to a large extent either in
exclusion or interaction regime. Hence, the elution of block copolymers in this case would
follow the elution behavior of the noncritical block.
Another unconventional combination of exclusion and adsorption interaction mechanisms
can be established by using different solvent systems for sample preparation and as the
mobile phase. The corresponding liquid chromatographic mode is termed as liquid chroma-
tography under limiting conditions of adsorption if the eluent is promoting adsorption while
the sample is prepared in a desorption promoting solvent. On the other hand, a desorption
promoting solvent as eluent and an adsorption promoting solvent as sample solvent render a
mode of liquid chromatography termed as liquid chromatography under limiting conditions
of desorption [62–77].

1.6 Multidimensional separation of complex polymers

Complex polymers exhibit more than one parameter of molecular heterogeneity that may
include chemical composition or functionality along with the inherent molar mass distribution.
Hence, the analysis of more than one distributed property requires the analysis of different dis-
tribution functions. The molecular heterogeneity of a copolymer AB with regard to molar mass
and chemical composition is presented in Fig. 1.6. The x-axis in Fig. 1.6 represents the molar
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and agility, and thought to crown her efforts by a notable feat, which
was no less than standing on her head on the top of the ladder, and
brandishing the two stilts, from which she had disengaged herself,
round about her, like the arms of a windmill. It required no great
skill to see that the old lady was very much offended with this last
performance, for when the little dish was carried to her, and the
ladder-dancer directed a beseeching look accompanied by an attitude
which seemed to imply that there were other feats yet in reserve, if
encouragement was held out, the patroness of the stair-head could
restrain herself no longer, but poured out a torrent partaking both of
objurgation and admonition.
“Ne’er-do-weel hussie,” and “vagrant gipsy,” were some of the
sharp missiles shot at the unsuspecting figurante, who, as little aware
of the meaning of all this “sharp-toothed violence,” as the bird is of
the mischief aimed at him by the fowler, sadly misapprehended its
import, and thinking it conveyed encouragement and approbation,
ducked her head in acknowledgment, while the thunder of the old
lady’s reprobation rolled about her in the most ceaseless rapidity of
vituperation.
“Ye’re a pretty ane indeed, to play sic antics afore ony body’s
house! Hae ye naebody to learn ye better manners that to rin up and
down a ladder like a squirrel, twisting and turning yoursel till my
banes are sair to look at you? Muckle fitter gin ye would read your
Bible, if as much grace be left to ye; or maybe a religious tract, to
begin wi’, for I doubt ye wad need preparation afore ye could drink at
the spring-head wi’ ony special profit.”
The last part was conveyed with a kind of smile of self-
approbation; for of all tasks, to reclaim a sinner is the most pleasing
and soothing to religious vanity;—so comfortable it is to be allowed
to scold on any terms, but doubly delightful, because it always
implies superiority. But the ladder-dancer and her attendant were
aware of no part of what was passing in the mind of the female
lecturer, and fully as ignorant of the eloquent address I have just
repeated; she only saw, in the gracious looks in which her feats were
condemned, an approval of her labours, for it passed her philosophy
to comprehend the ungodly qualities of standing on the head, or
whirling like a top. Again the ladder-dancer cringed and bowed to
her of the stair-head; and her male supporter, who acted as a kind of
pedestal to her elevation, bowed and grinned a little more grimly,
while the boy held out his plate to receive the results of all this
assiduity. But they could not command a single word of broad
English among them. Theirs only was the eloquence of nods and
grimaces; a monkey could have done as much, and in the present
humour of the old lady, would have been as much approved. The
ladder-dancer grew impatient, and seemed determined on an effort
to close her labours.
“Ah, Madame!” she exclaimed; “Madame” was repeated by the
man, and “Madame” was re-echoed by the boy.
“Nane o’ your nonsense wi’ me,” was the response from the stair-
head; “your madam’ing, and I dinna ken what mair havers. Ye
needna fash your head to stand there a’ day girning at me, and
making sic outlandish sport. I’m mair fule than you, that bides to
look at you; a fine tale they’d hae to tell that could say they saw me
here, idling my precious time on the like o’ you.”
She now whispered to one of the girls, who retired, and soon after
returned, giving her a small parcel, which she examined, and seemed
to say all was right. She beckoned the ladder-dancer, who slid down
with cat-like agility, and was instantly with her, standing a step
lower, in deference to the doughty dame.
“Here,” said she, with a gruff air, which was rather affected than
real, “tak these precious gifts,” handing her a bunch of religious
tracts. “See if ye canna find out your spiritual wants, and learn to
seek for the ‘Pearl of Price.’ My certie, but ye’re a weel-faured
hussie,” examining her more narrowly, “but your gaits are no that
commendable; but for a’ that, a mair broken ship has reached the
land.”
I could observe that she slipped a half-crown into the hand of the
Piedmontoise; and as she turned away to avoid thanks, an elderly
gentleman (perhaps her husband), who stood by, said in a low voice,
—
“That’s like yoursel, Darsie; your bark was aye waur than your bite,
ony day!”—Blackwood’s Magazine, 1826.
 THE ELDER’S DEATH-BED.

By Professor Wilson.

It was on a fierce and howling day that I was crossing the dreary
moor of Auchindown, on my way to the manse of that parish—a
solitary pedestrian. The snow, which had been incessantly falling for
a week past, was drifted into beautiful but dangerous wreaths, far
and wide, over the melancholy expanse; and the scene kept visibly
shifting before me, as the strong wind that blew from every point of
the compass struck the dazzling masses, and heaved them up and
down in endless transformation. There was something inspiriting in
the labour with which, in the buoyant strength of youth, I forced my
way through the storm; and I could not but enjoy those gleamings of
sunlight that ever and anon burst through some unexpected opening
in the sky, and gave a character of cheerfulness, and even warmth, to
the sides or summits of the stricken hills. Sometimes the wind
stopped of a sudden, and then the air was as silent as the snow—not
a murmur to be heard from spring or stream, now all frozen up over
those high moorlands. As the momentary cessations of the sharp
drift allowed my eyes to look onwards and around, I saw here and
there, up the little opening valleys, cottages just visible beneath the
black stems of their snow-covered clumps of trees, or beside some
small spot of green pasture kept open for the sheep. These
intimations of life and happiness came delightfully to me in the
midst of the desolation; and the barking of a dog, attending some
shepherd in his quest on the hill, put fresh vigour into my limbs,
telling me that, lonely as I seemed to be, I was surrounded by
cheerful, though unseen company, and that I was not the only
wanderer over the snows.
 As I walked along, my mind was insensibly filled with a crowd of
pleasant images of rural winter life, that helped me gladly onwards
over many miles of moor. I thought of the severe but cheerful labours
of the barn—the mending of farm-gear by the fireside—the wheel
turned by the foot of old age less for gain than as a thrifty pastime—
the skilful mother making “auld claes look amaist as weel’s the
new”—the ballad unconsciously listened to by the family all busy at
their own tasks round the singing maiden—the old traditionary tale,
told by some wayfarer hospitably housed till the storm should blow
by—the unexpected visit of neighbours on need or friendship—or the
footstep of lover undeterred by snow-drifts that have buried up his
flocks;—but above all, I thought of those hours of religious worship
that have not yet escaped from the domestic life of the peasantry of
Scotland—of the sound of psalms that the depth of the snow cannot
deaden to the ear of Him to whom they are chanted—and of that
sublime Sabbath-keeping which, on days too tempestuous for the
kirk, changes the cottage of the shepherd into the temple of God.
With such glad and peaceful images in my heart, I travelled along
that dreary moor, with the cutting wind in my face, and my feet
sinking in the snow, or sliding on the hard blue ice beneath it—as
cheerfully as I ever walked in the dewy warmth of a summer
morning, through fields of fragrance and of flowers. And now I could
discern, within half an hour’s walk, before me, the spire of the
church, close to which stood the manse of my aged friend and
benefactor. My heart burned within me as a sudden gleam of stormy
sunlight tipped it with fire; and I felt, at that moment, an
inexpressible sense of the sublimity of the character of that
grayheaded shepherd who had, for fifty years, abode in the
wilderness, keeping together his own happy little flock.
As I was ascending a knoll, I saw before me on horseback an old
man, with his long white hairs beaten against his face, who,
nevertheless, advanced with a calm countenance against the
hurricane. It was no other than my father, of whom I had been
thinking—for my father had I called him for many years, and for
many years my father had he truly been. My surprise at meeting him
on such a moor—on such a day—was but momentary, for I knew that
he was a shepherd who cared not for the winter’s wrath. As he
stopped to take my hand kindly into his, and to give his blessing to
his long-expected visitor, the wind fell calm—the whole face of the
sky was softened, and brightness, like a smile, went over the blushing
and crimson snow. The very elements seemed then to respect the
hoary head of fourscore; and after our first greeting was over, when I
looked around, in my affection, I felt how beautiful was winter.
“I am going,” said he, “to visit a man at the point of death; a man
whom you cannot have forgotten; whose head will be missed in the
kirk next Sabbath by all my congregation; a devout man, who feared
God all his days, and whom, on this awful trial, God will assuredly
remember. I am going, my son, to the Hazel Glen.”
I knew well in childhood that lonely farmhouse, so far off among
the beautiful wild green hills, and it was not likely that I had
forgotten the name of its possessor. For six years’ Sabbaths I had
seen the Elder in his accustomed place beneath the pulpit, and, with
a sort of solemn fear, had looked on his steadfast countenance during
sermon, psalm, and prayer. On returning to the scenes of my infancy,
I now met the pastor going to pray by his deathbed; and, with the
privilege which nature gives us to behold, even in their last
extremity, the loving and the beloved, I turned to accompany him to
the house of sorrow, resignation, and death.
And now, for the first time, I observed walking close to the feet of
his horse, a little boy of about ten years of age, who kept frequently
looking up in the pastor’s face, with his blue eyes bathed in tears. A
changeful expression of grief, hope, and despair, made almost pale
cheeks that otherwise were blooming in health and beauty; and I
recognised, in the small features and smooth forehead of childhood,
a resemblance to the aged man whom we understood was now lying
on his death-bed. “They had to send his grandson for me through the
snow, mere child as he is,” said the minister to me, looking tenderly
on the boy; “but love makes the young heart bold—and there is One
who tempers the wind to the shorn lamb.”
I again looked on the fearless child with his rosy cheeks, blue eyes,
and yellow hair, so unlike grief or sorrow, yet now sobbing aloud as if
his heart would break. “I do not fear but that my grandfather will yet
recover, as soon as the minister has said one single prayer by his
bedside. I had no hope, or little, as I was running by myself to the
manse over hill after hill, but I am full of hopes, now that we are
together; and oh! if God suffers my grandfather to recover, I will lie
awake all the long winter nights blessing Him for His mercy. I will
rise up in the middle of the darkness, and pray to Him in the cold on
my naked knees!” and here his voice was choked, while he kept his
eyes fixed, as if for consolation and encouragement, on the solemn
and pitying countenance of the kind-hearted pious old man.
We soon left the main road, and struck off through scenery that,
covered as it was with the bewildering snow, I sometimes dimly and
sometimes vividly remembered; our little guide keeping ever a short
distance before us, and with a sagacity like that of instinct, showing
us our course, of which no trace was visible, save occasionally his
own little footprints as he had been hurrying to the manse.
After crossing, for several miles, morass and frozen rivulet, and
drifted hollow, with here and there the top of a stone-wall peeping
through the snow, or the more visible circle of a sheep-bucht, we
descended into the Hazel-glen, and saw before us the solitary house
of the dying Elder.
A gleam of days gone by came suddenly over my soul. The last time
that I had been in this glen was on a day of June, fifteen years before,
—a holiday, the birthday of the king. A troop of laughing schoolboys,
headed by our benign pastor, we danced over the sunny braes, and
startled the linnets from their nests among the yellow broom.
Austere as seemed to us the Elder’s Sabbath face when sitting in the
kirk, we schoolboys knew that it had its week-day smiles, and we flew
on the wings of joy to our annual festival of curds and cream in the
farm-house of that little sylvan world. We rejoiced in the flowers and
the leaves of that long, that interminable summer day; its memory
was with our boyish hearts from June to June; and the sound of that
sweet name, “Hazel Glen,” often came upon us at our tasks, and
brought too brightly into the school-room the pastoral imagery of
that mirthful solitude.
As we now slowly approached the cottage through a deep snow-
drift, which the distress within had prevented the household from
removing, we saw peeping out from the door, brothers and sisters of
our little guide, who quickly disappeared, and then their mother
showed herself in their stead, expressing by her raised eyes, and
arms folded across her breast, how thankful she was to see at last the
pastor, beloved in joy and trusted in trouble.
 Soon as the venerable old man dismounted from his horse, our
active little guide led it away into the humble stable, and we entered
the cottage. Not a sound was heard but the ticking of the clock. The
matron, who had silently welcomed us at the door, led us, with
suppressed sighs and a face stained with weeping, into her father’s
sick room, which even in that time of sore distress was as orderly as
if health had blessed the house. I could not help remarking some old
china ornaments on the chimneypiece, and in the window was an
ever-blowing rose-tree, that almost touched the lowly roof, and
brightened that end of the apartment with its blossoms. There was
something tasteful in the simple furniture; and it seemed as if grief
could not deprive the hand of that matron of its careful elegance.
Sickness, almost hopeless sickness, lay there, surrounded with the
same cheerful and beautiful objects which health had loved; and she,
who had arranged and adorned the apartment in her happiness, still
kept it from disorder and decay in her sorrow.
With a gentle hand she drew the curtain of the bed, and there,
supported by pillows as white as the snow that lay without, reposed
the dying Elder. It was plain that the hand of God was upon him, and
that his days on the earth were numbered.
He greeted his minister with a faint smile, and a slight inclination
of the head—for his daughter had so raised him on the pillows, that
he was almost sitting up in his bed. It was easy to see that he knew
himself to be dying, and that his soul was prepared for the great
change; yet, along with the solemn resignation of a Christian who
had made his peace with God and his Saviour, there was blended on
his white and sunken countenance an expression of habitual
reverence for the minister of his faith; and I saw that he could not
have died in peace without that comforter to pray by his death-bed.
A few words sufficed to tell who was the stranger;—and the dying
man, blessing me by name, held out to me his cold shrivelled hand,
in token of recognition. I took my seat at a small distance from the
bedside, and left a closer station for those who were more dear. The
pastor sat down near his head; and, by the bed, leaning on it with
gentle hands, stood that matron, his daughter-in-law—a figure that
would have graced and sainted a higher dwelling, and whose native
beauty was now more touching in its grief. But religion upheld her
whom nature was bowing down. Not now for the first time were the
lessons taught by her father to be put into practice, for I saw that she
was clothed in deep mourning and she behaved like the daughter of a
man whose life had been not only irreproachable but lofty, with fear
and hope fighting desperately but silently in the core of her pure and
pious heart.
While we thus remained in silence, the beautiful boy, who, at the
risk of his life, had brought the minister of religion to the bedside of
his beloved grandfather, softly and cautiously opened the door, and
with the hoar-frost yet unmelted on his bright glistering ringlets,
walked up to the pillow, evidently no stranger there. He no longer
sobbed—he no longer wept—for hope had risen strongly within his
innocent heart, from the consciousness of love so fearlessly exerted,
and from the presence of the holy man in whose prayers he trusted,
as in the intercession of some superior and heavenly nature. There
he stood, still as an image in his grandfather’s eyes, that, in their
dimness, fell upon him with delight. Yet, happy as was the trusting
child, his heart was devoured by fear, and he looked as if one word
might stir up the flood of tears that had subsided in his heart. As he
crossed the dreary and dismal moors, he had thought of a corpse, a
shroud, and a grave; he had been in terror, lest death should strike in
his absence the old man, with whose gray hairs he had so often
played; but now he saw him alive, and felt that death was not able to
tear him away from the clasps, and links, and fetters of his
grandchild’s embracing love.
“If the storm do not abate,” said the sick man, after a pause, “it will
be hard for my friends to carry me over the drifts to the kirkyard.”
This sudden approach to the grave struck, as with a bar of ice, the
heart of the loving boy; and, with a long deep sigh, he fell down with
his face like ashes on the bed, while the old man’s palsied right hand
had just strength to lay itself upon his head. “Blessed be thou, my
little Jamie, even for His own name’s sake who died for us on the
tree!” The mother, without terror, but with an averted face, lifted up
her loving-hearted boy, now in a dead fainting-fit, and carried him
into an adjoining room, where he soon revived. But that child and
the old man were not to be separated. In vain he was asked to go to
his brothers and sisters;—pale, breathless, and shivering, he took his
place as before, with eyes fixed on his grandfather’s face, but neither
weeping nor uttering a word. Terror had frozen up the blood of his
heart; but his were now the only dry eyes in the room; and the pastor
himself wept—albeit the grief of fourscore is seldom vented in tears.
“God has been gracious to me, a sinner,” said the dying man.
“During thirty years that I have been an elder in your kirk, never
have I missed sitting there one Sabbath. When the mother of my
children was taken from me—it was on a Tuesday she died, and on
Saturday she was buried—we stood together when my Alice was let
down into the narrow house made for all living; on the Sabbath I
joined in the public worship of God: she commanded me to do so the
night before she went away. I could not join in the psalm that
Sabbath, for her voice was not in the throng. Her grave was covered
up, and grass and flowers grew there; so was my heart; but thou,
whom, through the blood of Christ, I hope to see this night in
Paradise, knowest that, from that hour to this day, never have I
forgotten thee!”
The old man ceased speaking, and his grandchild, now able to
endure the scene (for strong passion is its own support), glided softly
to a little table, and bringing a cup in which a cordial had been
mixed, held it in his small soft hands to his grandfather’s lips. He
drank, and then said, “Come closer to me, Jamie, and kiss me for
thine own and thy father’s sake;” and as the child fondly pressed his
rosy lips on those of his grandfather, so white and withered, the tears
fell over all the old man’s face, and then trickled down on the golden
head of the child, at last sobbing in his bosom.
“Jamie, thy own father has forgotten thee in thy infancy, and me in
my old age; but, Jamie, forget not thou thy father nor thy mother, for
that thou knowest and feelest is the commandment of God.”
The broken-hearted boy could give no reply. He had gradually
stolen closer and closer unto the old loving man, and now was lying,
worn out with sorrow, drenched and dissolved in tears, in his
grandfather’s bosom. His mother had sunk down on her knees and
hid her face with her hands. “Oh! if my husband knew but of this—he
would never, never desert his dying father!” and I now knew that the
Elder was praying on his death-bed for a disobedient and wicked
son.
At this affecting time the minister took the family Bible on his
knees, and said, “Let us sing to the praise and glory of God, part of
the fifteenth psalm;” and he read, with a tremulous and broken voice,
those beautiful verses:—
“Within thy tabernacle, Lord,
Who shall abide with thee?
And in Thy high and holy hill
Who shall a dweller be?
The man that walketh uprightly,
And worketh righteousness,
And as he thinketh in his heart,
So doth he truth express.”

The small congregation sang the noble hymn of the psalmist to

“plaintiff Martyrs, worthy of the name.” The dying man himself, ever
and anon, joined in the holy music; and when it feebly died away on
his quivering lips, he continued still to follow the tune with the
motion of his withered hand, and eyes devoutly and humbly lifted up
to heaven. Nor was the sweet voice of his loving grandchild unheard;
as if the strong fit of deadly passion had dissolved in the music, he
sang with a sweet and silvery voice, that, to a passer-by, had seemed
that of perfect happiness—a hymn sung in joy upon its knees by
gladsome childhood before it flew out among the green hills, to quiet
labour or gleesome play. As that sweetest voice came from the bosom
of the old man, where the singer lay in affection, and blended with
his own so tremulous, never had I felt so affectingly brought before
me the beginning and the end of life, the cradle and the grave.
Ere the psalm was yet over, the door was opened, and a tall fine-
looking man entered, but with a lowering and dark countenance,
seemingly in sorrow, in misery, and remorse. Agitated, confounded,
and awe-struck by the melancholy and dirge-like music, he sat down
on a chair, and looked with a ghastly face towards his father’s death-
bed. When the psalm ceased, the Elder said with a solemn voice, “My
son, thou art come in time to receive thy father’s blessing. May the
remembrance of what will happen in this room before the morning
again shine over the Hazel Glen win thee from the error of thy ways!
Thou art here, to witness the mercy of thy God and thy Saviour,
whom thou hast forgotten.”
The minister looked, if not with a stern, yet with an upbraiding
countenance, on the young man, who had not recovered his speech,
and said, “William! for three years past your shadow has not
darkened the door of the house of God. They who fear not the
thunder may tremble at the still small voice; now is the hour for
repentance, that your father’s spirit may carry up to heaven tidings of
a contrite soul saved from the company of sinners!”
The young man, with much effort, advanced to the bedside, and at
last found voice to say, “Father, I am not without the affections of
nature, and I hurried home as soon as I heard that the minister had
been seen riding towards our house. I hope that you will yet recover,
and if I have ever made you unhappy, I ask your forgiveness; for
though I may not think as you do on matters of religion, I have a
human heart. Father! I may have been unkind, but I am not cruel. I
ask your forgiveness.”
“Come nearer to me, William; kneel down by the bedside, and let
my hand find the head of my beloved son—for blindness is coming
fast upon me. Thou wert my first-born, and thou art my only living
son. All thy brothers and sisters are lying in the kirkyard, beside her
whose sweet face thine own, William, did once so much resemble.
Long wert thou the joy, the pride of my soul—ay, too much the pride,
for there was not in all the parish such a man, such a son, as my own
William. If thy heart has since been changed, God may inspire it
again with right thoughts. Could I die for thy sake—could I purchase
thy salvation with the outpouring of thy father’s blood—but this the
Son of God has done for thee, who hast denied Him! I have sorely
wept for thee—ay, William, when there was none near me—even as
David wept for Absalom, for thee, my son, my son!”
A long deep groan was the only reply; but the whole body of the
kneeling man was convulsed; and it was easy to see his sufferings, his
contrition, his remorse, and his despair. The pastor said, with a
sterner voice and austerer countenance than were natural to him,
“Know you whose hand is now lying on your rebellious head? But
what signifies the word father to him who has denied God, the Father
of us all?”—“Oh! press him not so hardly,” said the weeping wife,
coming forward from a dark corner of the room, where she had tried
to conceal herself in grief, fear, and shame. “Spare, oh! spare my
husband—he has ever been kind to me;” and with that she knelt
down beside him, with her long, soft, white arms mournfully and
affectionately laid across his neck. “Go thou, likewise, my sweet little
Jamie,” said the Elder, “go even out of my bosom, and kneel down
beside thy father and thy mother, so that I may bless you all at once,
and with one yearning prayer.” The child did as that solemn voice
commanded, and knelt down somewhat timidly by his father’s side;
nor did that unhappy man decline encircling with his arm the child
too much neglected, but still dear to him as his own blood, in spite of
the deadening and debasing influence of infidelity.
“Put the Word of God into the hands of my son, and let him read
aloud to his dying father the 25th, 26th, and 27th verses of the
eleventh chapter of the Gospel according to St John.” The pastor
went up to the kneelers, and, with a voice of pity, condolence, and
pardon, said, “There was a time when none, William, could read the
Scriptures better than couldst thou—can it be that the son of my
friend hath forgotten the lessons of his youth?” He had not forgotten
them; there was no need for the repentant sinner to lift up his eyes
from the bedside. The sacred stream of the Gospel had worn a
channel in his heart, and the waters were again flowing. With a
choked voice he said, “Jesus said unto her, I am the Resurrection and
the Life: he that believeth in me, though he were dead, yet shall he
live: and whosoever liveth and believeth in me, shall never die.
Believest thou this? She saith unto him, Yea, Lord; I believe that thou
art the Christ, the Son of God, which should come into the world.”
“That is not an unbeliever’s voice,” said the dying man
triumphantly; “nor, William, hast thou an unbeliever’s heart. Say
that thou believest in what thou hast now read, and thy father will
die happy!”—“I do believe; and as thou forgivest me, so may I be
forgiven by my Father who is in heaven.”
The Elder seemed like a man suddenly inspired with a new life. His
faded eyes kindled—his pale cheeks glowed—his palsied hands
seemed to wax strong—and his voice was clear as that of manhood in
its prime. “Into Thy hands, O God, I commit my spirit!”—and so
saying, he gently sank back on his pillow; and I thought I heard a
sigh. There was then a long deep silence, and the father, and mother,
and child rose from their knees. The eyes of us all were turned
towards the white placid face of the figure now stretched in
everlasting rest; and without lamentations, save the silent
lamentations of the resigned soul, we stood around the “Death-bed
of the Elder.”
 A HIGHLAND FEUD.

By Sir Walter Scott.

The principal possessors of the Hebrides were originally of the

name of MacDonald, the whole being under the government of a
succession of chiefs, who bore the name of Donald of the Isles, and
were possessed of authority almost independent of the kings of
Scotland. But this great family becoming divided into two or three
branches, other chiefs settled in some of the islands, and disputed
the property of the original proprietors. Thus, the MacLeods, a
powerful and numerous clan, who had extensive estates on the
mainland, made themselves masters, at a very early period, of a great
part of the large island of Skye, seized upon much of the Long Island,
as the isles of Lewis and Harris are called, and fought fiercely with
the MacDonalds and other tribes of the islands. The following is an
example of the mode in which these feuds were conducted:—
About the end of the sixteenth century, a boat, manned by one or
two of the MacLeods, landed in Eigg, a small island peopled by the
MacDonalds. They were at first hospitably received; but having been
guilty of some incivility to the young women of the island, it was so
much resented by the inhabitants, that they tied the MacLeods hand
and foot, and putting them on board of their own boat, towed it to
the sea, and set it adrift, leaving the wretched men, bound as they
were, to perish by famine, or by the winds and waves, as chance
should determine. But fate so ordered it, that a boat belonging to the
Laird of MacLeod fell in with that which had the captives on board,
and brought them in safety to the Laird’s castle of Dunvegan, in
Skye, where they complained of the injury which they had sustained
from the MacDonalds of Eigg. MacLeod, in great rage, put to sea
with his galleys, manned by a large body of his people, which the
men of Eigg could not entertain any rational hope of resisting.
Learning that their incensed enemy was approaching with superior
forces, and deep vows of revenge, the inhabitants, who knew they
had no mercy to expect at MacLeod’s hands, resolved, as the best
chance of safety in their power, to conceal themselves in a large
cavern on the sea-shore.
This place was particularly well-calculated for that purpose. The
entrance resembles that of a fox-earth, being an opening so small
that a man cannot enter save by creeping on hands and knees. A rill
of water falls from the top of the rock, and serves, or rather served at
the period we speak of, wholly to conceal the aperture. A stranger,
even when apprised of the existence of such a cave, would find the
greatest difficulty in discovering the entrance. Within, the cavern
rises to a great height, and the floor is covered with white dry sand. It
is extensive enough to contain a great number of people. The whole
inhabitants of Eigg, who, with their wives and families, amounted to
nearly two hundred souls, took refuge within its precincts.
MacLeod arrived with his armament, and landed on the island, but
could discover no one on whom to wreak his vengeance—all was
desert. The MacLeods destroyed the huts of the islanders, and
plundered what property they could discover; but the vengeance of
the chieftain could not be satisfied with such petty injuries. He knew
that the inhabitants must either have fled in their boats to one of the
islands possessed by the MacDonalds, or that they must be concealed
somewhere in Eigg. After making a strict but unsuccessful search for
two days, MacLeod had appointed the third to leave his anchorage,
when, in the gray of the morning, one of the seamen beheld, from the
deck of his galley, the figure of a man on the island. This was a spy
whom the MacDonalds, impatient of their confinement in the cavern,
had imprudently sent out to see whether MacLeod had retired or no.
The poor fellow, when he saw himself discovered, endeavoured, by
doubling after the manner of a hare or fox, to obliterate the track of
his footsteps, and prevent its being discovered where he had re-
entered the cavern. But all his art was in vain; the invaders again
landed, and tracked him to the entrance of the cavern.
MacLeod then summoned those who were within it, and called
upon them to deliver the individuals who had maltreated his men, to
be disposed of at his pleasure. The MacDonalds, still confident in the
strength of their fastness, which no assailant could enter but on
hands and knees, refused to surrender their clansmen.
MacLeod then commenced a dreadful work of indiscriminate
vengeance. He caused his people, by means of a ditch cut above the
top of the rock, to turn away the stream of water which fell over the
entrance of the precipice. This being done, the MacLeods collected
all the combustibles which could be found on the island, particularly
quantities of dry heather, piled them up against the aperture, and
maintained an immense fire for many hours, until the smoke,
penetrating into the inmost recesses of the cavern, stifled to death
every creature within. There is no doubt of the truth of this story,
dreadful as it is. The cavern is often visited by strangers; and I have
myself seen the place, where the bones of the murdered MacDonalds
still remain, lying as thick on the floor of the cave as in the charnel-
house of a church.
 THE RESURRECTION MEN.

By D. M. Moir, M.D.
How then was the Devil drest?
He was in his Sunday’s best;
His coat was red, and his breeches were blue,
With a hole behind, where his tail came through.

Over the hill, and over the dale,

And he went over the plain:
And backward and forward he switched his tail,
As a gentleman switches his cane.
Coleridge.

About this time[8] there arose a great sough and surmise that some
loons were playing false with the kirkyard, howking up the bodies
from their damp graves, and hurling them away to the college.
Words canna describe the fear, and the dool, and the misery it
caused. All flocked to the kirk yett; and the friends of the newly
buried stood by the mools, which were yet dark, and the brown,
newly-cast divots, that had not yet ta’en root, looking with mournful
faces, to descry any tokens of sinking in.
8. See ante, “Benjie’s Christening,” page 214.
I’ll never forget it. I was standing by when three young lads took
shools, and, lifting up the truff, proceeded to howk down to the
coffin, wherein they had laid the gray hairs of their mother. They
looked wild and bewildered like, and the glance of their een was like
that of folk out of a mad-house; and none dared in the world to have
spoken to them. They didna even speak to ane anither; but wrought
on wi’ a great hurry till the spades struck on the coffin-lid—which
was broken. The dead-claithes were there huddled a’thegither in a
nook, but the dead was gane. I took haud o’ Willie Walker’s arm, and
looked down. There was a cauld sweat all ower me;—losh me! but I
was terribly frighted and eerie. Three mair graves were opened, and
a’ just alike, save and except that of a wee unkirstened wean, which
was aff bodily, coffin and a’.
There was a burst of righteous indignation throughout the parish;
nor without reason. Tell me that doctors and graduates maun hae the
dead; but tell it not to Mansie Wauch, that our hearts maun be
trampled in the mire of scorn, and our best feelings laughed at, in
order that a bruise may be properly plaistered up, or a sair head
cured. Verily, the remedy is waur than the disease.
But what remead? It was to watch in the session-house, with
loaded guns, night about, three at a time. I never likit to gang into
the kirkyard after darkening, let-a-be to sit there through a lang
winter night, windy and rainy, it may be, wi’ nane but the dead
around us. Save us! it was an unco thought, and garred a’ my flesh
creep; but the cause was gude,—my spirit was roused, and I was
determined no to be dauntoned.
I counted and counted, but the dread day at length came, and I
was summonsed. All the leivelang afternoon, when ca’ing the needle
upon the brod, I tried to whistle Jenny Nettles, Niel Gow, and ither
funny tunes, and whiles crooned to mysel between hands; but my
consternation was visible, and a’ wadna do.
It was in November, and the cauld glimmering sun sank behind
the Pentlands. The trees had been shorn of their frail leaves; and the
misty night was closing fast in upon the dull and short day; but the
candles glittered at the shop windows, and leery-light-the-lamps was
brushing about wi’ his ladder in his oxter, and bleezing flamboy
sparking out behind him. I felt a kind of qualm of faintness and
down-sinking about my heart and stomach, to the dispelling of which
I took a thimbleful of spirits, and, tying my red comforter about my
neck, I marched briskly to the session-house. A neighbour (Andrew
Goldie, the pensioner) lent me his piece, and loaded it to me. He took
tent that it was only half-cock, and I wrapped a napkin round the
dog-head, for it was raining. No being acquaint wi’ guns, I keepit the
muzzle aye awa frae me; as it is every man’s duty no to throw his
precious life into jeopardy.
 A furm was set before the session-house fire, which bleezed
brightly, nor had I ony thought that such an unearthly place could
have been made to look half so comfortable, either by coal or candle;
so my speerits rose up as if a weight had been ta’en aff them, and I
wondered in my bravery, that a man like me could be afeard of
onything. Nobody was there but a touzy, ragged, halflins callant of
thirteen (for I speired his age), wi’ a desperate dirty face, and lang
carroty hair, tearing a speldrin wi’ his teeth, which lookit lang and
sharp eneugh, and throwing the skin and lugs intil the fire.
We sat for amaist an hour thegither, cracking the best way we
could in sic a place; nor was onybody mair likely to cast up. The night
was now pit-mirk; the wind soughed amid the headstanes and
railings of the gentry (for we maun a’ dee); and the black corbies in
the steeple-holes cackled and crawed in a fearsome manner. A’ at
ance we heard a lonesome sound; and my heart began to play pit-pat
—my skin grew a’ rough, like a poukit chicken—and I felt as if I didna
ken what was the matter with me. It was only a false alarm, however,
being the warning of the clock; and in a minute or twa thereafter the
bell struck ten. Oh, but it was a lonesome and dreary sound! Every
chap gaed through my breast like the dunt of a forehammer.
Then up and spak the red headed laddie: “It’s no fair; anither
should hae come by this time. I wad rin awa hame, only I’m
frightened to gang out my lane. Do ye think the doup o’ that candle
wad carry in my cap?”
“Na, na, lad; we maun bide here, as we are here now. Leave me
alane! Lord save us! and the yett lockit, and the bethrel sleepin’ wi’
the key in his breek-pouches! We canna win out now, though we
would,” answered I, trying to look brave, though half frightened out
of my seven senses. “Sit down, sit down; I’ve baith whisky and porter
wi’ me. Hae, man, there’s a cauker to keep your heart warm; and set
down that bottle,” quoth I, wiping the sawdust aff it with my hand,
“to get a toast; I’se warrant it for Deacon Jaffrey’s best brown stout.”
The wind blew higher, and like a hurricane; the rain began to fall
in perfect spouts; the auld kirk rumbled, and rowed, and made a sad
soughing; and the bourtree tree behind the house, where auld
Cockburn, that cuttit his throat, was buried, creakit and crazed in a
frightful manner; but as to the roaring of the troubled waters, and
the bumming in the lum-head, they were past a’ power of
description. To make bad worse, just in the heart of the brattle, the
grating sound of the yett turning on its rusty hinges was but too
plainly heard. What was to be done? I thought of our baith running
away; and then of our locking oursels in, and firing through the door;
but wha was to pull the trigger?
Gudeness watch ower us! I tremble yet when I think on’t. We were
perfectly between the deil and the deep sea—either to stand and fire
our gun, or rin and be shot at. It was really a hang choice. As I stood
swithering and shaking, the laddie ran to the door, and thrawing
round the key, clapped his back till’t. Oh! how I lookit at him, as he
stude, for a gliff, like a magpie hearkening wi’ his lug cockit up, or
rather like a terrier watching a rotten.
“They’re coming! they’re coming!” he cried out; “cock the piece, ye
sumph,” while the red hair rose up from his pow like feathers;
“they’re coming, I hear them tramping on the gravel!” Out he
stretched his arms against the wall, and brizzed his back against the
door like mad; as if he had been Samson pushing over the pillars in
the house of Dagon. “For the Lord’s sake, prime the gun,” he cried
out, “or our throats will be cut frae lug to lug, before we can say Jack
Robinson! See that there’s priming in the pan!”
I did the best I could; but my hale strength could hardly lift up the
piece, which waggled to and fro like a cock’s tail on a rainy day; my
knees knockit against ane anither, and though I was resigned to dee
—I trust I was resigned to dee—’od, but it was a frightfu’ thing to be
out of ane’s bed, and to be murdered in an auld session-house, at the
dead hour of night, by unyearthly resurrection-men—or rather let me
call them devils incarnate—wrapt up in dreadnoughts, wi’ blackit
faces, pistols, big sticks, and other deadly weapons.
A snuff-snuffing was heard; and through below the door I saw a
pair of glancing black een. ’Od, but my heart nearly loupit aff the bit
—a snouff and a gur—gurring, and ower a’ the plain tramp of a man’s
heavy tackets and cuddy-heels amang the gravel. Then cam a great
slap like thunder on the wall; and the laddie quitting his grip, fell
down, crying, “Fire, fire!—murder! holy murder!”
“Wha’s there?” growled a deep rough voice; “open—I’m a friend.”
I tried to speak, but could not; something like a halfpenny roll was
sticking in my throat, so I tried to cough it up, but it wadna come.
“Gie the pass-word, then,” said the laddie, staring as if his een wad
loupen out; “gie the pass-word!”
First cam a loud whussle, and then “Copmahagen,” answered the
voice. Oh! what a relief! The laddie started up like ane crazy wi’ joy.
“Ou! ou!” cried he, thrawing round the key, and rubbing his hands,
“by jingo! it’s the bethrel—it’s the bethrel—it’s auld Isaac himsel!”
First rushed in the dog, and then Isaac, wi’ his glazed hat, slouched
ower his brow, and his horn bowet glimmering by his knee. “Has the
French landit, do ye think? Losh keep us a’!” said he, wi’ a smile on
his half-idiot face (for he was a kind of a sort of a natural, wi’ an
infirmity in his leg). “’Od sauf us, man, put by your gun. Ye dinna
mean to shoot me, do ye? What are ye aboot here wi’ the door lockit?
I just keppit four resurrectioners louping ower the wa’.”
“Gude guide us!” I said, taking a long breath to drive the blude frae
my heart, and something relieved by Isaac’s company. “Come now,
Isaac, ye’re just giein’ us a fright. Isn’t that true, Isaac?”
“Yes, I’m joking,—and what for no? But they might have been, for
onything ye wad hae hindered them to the contrair, I’m thinking. Na,
na, ye maunna lock the door; that’s no fair play.”
When the door was put ajee, and the furm set fornent the fire, I
gied Isaac a dram to keep his heart up on sic a cauld, stormy night.
’Od, but he was a droll fallow, Isaac. He sung and leuch as if he had
been boozing in Lucky Tamson’s, wi’ some of his drucken cronies.
Fient a hair cared he about auld kirks, or kirkyards, or vouts, or
through-stanes, or dead folk in their winding-sheets, wi’ the wet
grass growing ower them; and at last I began to brighten up a wee
mysel; so when he had gone ower a good few funny stories, I said to
him, quoth I, “Mony folk, I daresay, mak mair noise about their
sitting up in a kirkyard than it’s a’ worth. There’s naething here to
harm us.”
“I beg to differ wi’ ye there,” answered Isaac, taking out his horn
mull from his coat pouch, and tapping on the lid in a queer style—“I
could gie anither version of that story. Did ye no ken of three young
doctors—Eirish students—alang wi’ some resurrectioners, as waff
and wild as themselves, firing shottie for shottie wi’ the guard at
Kirkmabreck, and lodging three slugs in ane o’ their backs, forbye
firing a ramrod through anither ane’s hat?”