Molecular Characterization of Polymers

A Fundamental Guide : A Fundamental

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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
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