Figure 3.7 Molecular weight distributions. (From Introduction to Polymer Chemistry by R.

Sey-
mour, McGraw-Hill, New York, 1971. Used with permission.)

Typical techniques for molecular weight determination are given in Table 3.4. The
most popular techniques will be considered briefly.
All classic molecular weight determination methods require the polymer to be in
solution. To minimize polymer–polymer interactions, solutions equal to and less than 1
g of polymer to 100 mL of solution are utilized. To further minimize solute interactions,
extrapolation of the measurements to infinite dilution is normally practiced.
When the exponent a in the Mark-Houwink equation is equal to 1, the average
molecular weight obtained by viscosity measurements (Mv) is equal to Mw. However,
since typical values of a are 0.5 to 0.8, the value Mw is usually greater than Mv. Since
viscometry does not yield absolute values of M as is the case with other techniques, one
must plot [␩] against known values of M and determine the constants K and a in the
Mark-Houwink equation. Some of these values are available in the Polymer Handbook
(Burrell, 1974), and simple comparative effluent times or melt indices are often sufficient
for comparative purposes and quality control where K and a are known.
For polydisperse polymer samples, molecular weight values determined from colli-
gative properties (3.6–3.8), light scattering photometry (3.10), and the appropriate data
treatment of ultracentrifugation (3.11) are referred to as “absolute molecular weights,”
while those determined from gel permeation chromatography (GPC) (3.5) and viscometry
(3.13) are referred to as relative molecular weights. An absolute molecular weight is one
that can be determined experimentally and where the molecular weight can be related,
through basic equations, to the parameter(s) measured. GPC and viscometry require cali-
bration employing polymers of known molecular weight determined from an absolute
molecular weight technique.

3.4 FRACTIONATION OF POLYDISPERSE SYSTEMS

The data plotted in Fig. 3.7 were obtained by the fractionation of a polydisperse polymer.
Prior to the introduction of GPC, polydisperse polymers were fractionated by the addition

Copyright © 2003 by Marcel Dekker, Inc. All Rights Reserved.

Table 3.4 Typical Molecular Weight Determination Methodsa
Type of mol. wt. Applicable wt.
Method average range Other information
—
Light scattering Mw To ∞ Can also give shape
—
Membrane osmometry Mn 2  104 to 2  106
—
Vapor phase osmometry Mn To 40,000
—
Electron and X-ray microscopy Mn,w,z 102 to ∞ Shape, distribution
—
Isopiestic method (isothermal Mn To 20,000
distillation)
—
Ebulliometry (boiling point Mn To 40,000
elevation)
—
Cryoscopy (melting point Mn To 50,000
depression)
—
End-group analysis Mn To 20,000
—
Osmodialysis Mn 500–25,000
Centrifugation
—
Sedimentation equilibrium Mz To ∞
—
Archibald modification Mz,w To ∞
—
Trautman’s method Mw To ∞
Sedimentation velocity Gives a real To ∞
M only for
monodisperse
systems
Chromatography Calibrated To ∞ Mol. wt. distribution
SAXS Mw
Mass spectroscopy To 106
Viscometry Calibrated To ∞
Coupled chromatography-LS To ∞ Mol. wt. distribution,
shape, Mw, Mn
a
“To ∞” means that the molecular weight of the largest particles soluble in a suitable solvent can be determined
in theory.

of a nonsolvent to a polymer solution, by cooling a solution of polymer, solvent evapora-

tion, zone melting, extraction, diffusion, or centrifugation. The molecular weight of the
fractions may be determined by any of the classic techniques previously mentioned and
discussed subsequently in this chapter.
The least sophisticated but most convenient technique is fractional precipitation,
which is dependent on the slight change in the solubility parameter with molecular weight.
Thus, when a small amount of miscible nonsolvent is added to a polymer solution at a
constant temperature, the product with the highest molecular weight precipitates. This
procedure may be repeated after the precipitate is removed. These fractions may also be
redissolved and again fractionally precipitated.
For example, isopropyl alcohol or methanol may be added dropwise to a solution
of polystyrene in benzene until the solution becomes turbid. It is preferable to heat this
solution and allow it to cool before removing the first and subsequent fractions. Extraction
of a polymer in a Soxhlet-type apparatus in which fractions are removed at specific time
intervals may also be used as a fractionation procedure.

Copyright © 2003 by Marcel Dekker, Inc. All Rights Reserved.

844 Modern Physical Chemistry

Mm 
 4  10   32 10   25 10    675 10   1.132 10
8 6 6 6 9

106000 106000
Mm  10.6 103 lb / mol

i N i M3i
(iii) Mz 
i N i M2i

Mz 
4  10000   8  2000    1  5000    3  15000  
3 3 3 3

4  10000   8  2000    1  5000    3  15000  

2 2 2 2

Mz 
4 10   64 10   125 10   10125 10   1.4314 10
12 9 9 9 13

4  10   32 10   25 10    675 10 

8 6 6
1.132  10
6 9

Mz  12.64 103 lb / mol

Calculates values shows that

   
Mz 12.64 103 lb / mol  Mm 10.6 103 lb / mol  Mn 4 103 lb / mol  
13.8 MOLECULAR MASS DETERMINATION METHODS
Osmometry, viscometry, light
scattering and sedimentation methods are
commonly used methods for determination of
molar mass of a polymer. Some methods are
given here in detail.

13.8.1 Viscosity Method

Viscosity of a fluid is a fundamental
property which has been described in section
11.11. Viscosity measurements can be used for
determination of molar mass of a polymer.
Ostwald viscometer is the simplest apparatus
which is used for measurement of viscosity of
a solution. Ostwald viscometer consists of a
bulb B with markings x1 and x2 attached to a
capillary tube C and a reservoir D as shown in
Fig. 13.13. A liquid is introduced from C and Fig. 13.13 Ostwald viscometer
sucked from B side. Then time of flow of liquid
from x1 to x2 is recorded. The value of relative viscosity can be measured using
following formula.
  t
rel   
 0  0 t0
Where,
 Polymer Chemistry 845

  viscosity of solution
0 = viscosity of solvent
t = time of flow of solution
t 0  time of flow of solvent
 = density of solution
0 = density of solvent

Time flow can be measured by Ostwald viscometer while densities can be

determined by filling gravity bottle. Specific viscosity is obtained from relative
viscosity using the following relation.
sp  rel  1

The specific viscosity is converted into reduced viscosity by the relation

sp
red 
C
Where, C is the concentration in gmL-1.
 sp
is plotted as a
C
function of concentration of
polymer as shown in Fig.
13.14. The intercept of the
plot is viscosity of the solution
at infinite dilution and it is
called intrinsic viscosity i.e.
 sp
Intrinsic viscosity =    Limc 0
C
The relation between
  and molar mass of a
polymer is given by the
equation
   KM 
Fig. 13.14 Plot of ηsp/c as a function of
Where,  and K are
concentration.
empirical constants and M is
the molar mass of polymer. The above equation can be used for determination of
molar mass of polymer for which K and  are known.

13.8.2 Sedimentation
Let us consider a particle of polymer in solution which moves under the
influence of gravitational field. The falling particle having mass ms experiences a
centrifugal force which is equal to  2 rm and buoyancy force. The net force acting on
the particle can be written as
846 Modern Physical Chemistry

Net force = centrifugal force – buoyancy force

mv 2
   2 rms
r
m  r 
2

   2 rms
r
mr 2 2
   2 rms
r
 mr 2   2 rms (13.105)

Where, ω is the angular velocity of the rotor in radius per second, r is the
distance from centre of rotation of particle.

As we know that ms   v , by putting this value in equation (13.105),

 mr 2   2 r  v
So, net force will be equal to frictional force

 dr 
f    mr 2   2 r  v
 dt 
 dr  v
f    mr 2   2 r  m  
 dt  m
 dr 
f    mr 2   2 r  mv
 dt 
 dr 

f     2 mr 1   v
 dt 


 dr   mr 1   v
2

 
 dt  f
dr
dt  
N Am 1   v 
 2r NA f

Where, v is the partial specific volume, dr is the sedimentation velocity.

dt
dr
Left hand side is replaced by S because the term dt is the sedimentation
ω2 r
coefficient (S). NA is Avogadro’s number.
 Polymer Chemistry 847

S

M 1 v 
NA f
SN A f
M (13.106)
1 v
According to Stoke’s law frictional coefficient of a spherical particle is

f  6 rs
By putting value of f in equation (13.106), we get

SN A  6 rs 
M (13.107)
1 v
According to Stoke’s Einstein equation, we know
k BT
D
6 rs
k BT
6 rs 
D
Where, kB is the Boltzmann constant, D is the diffusion coefficient and T is
the absolute temperature.

By putting value of 6 rs in equation (13.107), we get

SN A k BT
M

D 1 v 
SRT
M

D 1 v 
In the above equation, unknown quantities are v , D and S. v and D can be
measured by some separate experiments. The sedimentation coefficient can be
determined by the following ways.

dr
S dt
 r 2

1 dr
Sdt 
2 r
By integrating above equation within limits
848 Modern Physical Chemistry

t r
1 1
S  dt   r dr
0
 2
r0

1
S t  0 
r
ln r r
 2 0

1
St   ln r  ln r0  (13.108)
2

ln  r 
St   0 
r
2

ln  r 
1
S
t 2
 r0 
By rearranging equation (13.108), we get

St 2  ln r  ln r0

ln r  St 2  ln r0 (13.109)

Equation (13.109) is an equation of straight line in intercept form as shown

in Fig. 13.15 with slope S . From the slope we can determined the value of
2

sedimentation coefficient.

Fig. 13.15 Plot of lnr versus t

13.8.3 Osmometry
Vapour phase and membrane osmometry are two principal types of osmometry
used so far to measure number average molecular weight.
 Polymer Chemistry 849

(i) Vapour Phase Osmometry

Principle
Addition of solute molecules decreases the vapour pressure of a liquid.
Decrease in vapour pressure depends upon number of solute molecules.
Construction and Working
Vapour phase osmometer is shown in Fig. 13.16. Two thermistors are
connected to a Wheatstone bridge. Solvent drops were placed in vicinity of both
thermistors by using syringes. As both wires are in proximity of same medium as
rate of evaporation is same at both wires, so no temperature difference is sensed
between both wires and a balance point is established. Now place drop of solution at
one thermistor and drop of solvent at the other. Now thermistors detect a
temperature difference as rate of evaporation at both ends is unequal. This
temperature difference is recorded in terms of potential difference by Wheatstone
bridge.
Calculation of Molecular Weight
Output voltage ΔV is related to molecular weight M by following equation
V K
  KBC (13.110)
C Mn
K is a calibration constant. Firstly value of K is determined by using polymer
of low and known molecular mass having less polydispersity. Then that value of K is
used to determine molecular mass. Voltage difference is measured for solutions of
different concentrations, then ΔV/C is plotted against concentration as shown in Fig.
13.16. Value of slope is equal to KB, while value of intercept is equal to K/ Mn . K is
known already by calibration, thus Mn is determined.
Advantages: Quick determination of molecular mass along with use of small
amount of polymer is two advantages of this method.
Disadvantages: Only low molecular weight possessing polymers (~30,000) can be
successfully measured by it.

Fig. 3.13 A sketch of a vapor pressure osmometer. Fig. 13.16 Working of vapour phase osmometry Fig. 13.17 Plot of ΔV/C versus C
(Courtesy of Hewlett-Packard Company.)
Problem
Insulin, a hormone that regulates carbohydrate metabolism in the blood, was isolated from a pig. A 0.200-g sample of insulin was
dissolved in 25.0 mL of water, and at 30 oC the osmotic pressure of the solution was found to be 26.1 torr. What is the molecular weight
of the insulin?
Solution: M = 5800
NOTE : This is an “apparent” molecular weight since it is for a single concentration and is not extrapolated to zero concentration.
850 Modern Physical Chemistry

(ii) Membrane Osmometry

Principle: Osmotic pressure of solvent decreases by addition of solute molecules.
Decrease in osmotic pressure depends upon number of solute molecules and related
to molar mass of polymer.
Types: Static, dynamic and high speed membrane osmometers are three kinds of
membrane osmometers which are normally use.
(I) Static Membrane Osmometer: In this osmometer, both chambers are
separated by a membrane and they are individually connected to small capillaries as
shown in Fig. 13.18. Initially both chambers contain solvent, thus there is no net
movement of molecules from one chamber to other. So level of liquid in both
capillaries is same as shown in Fig. 13.18 (a). Later solution is placed in one chamber
while other contains solvent. Now chemical and osmotic potential of both chambers
becomes unequal as shown in Fig. 13.18 (b). Now, solvent molecules move from
chamber of low osmotic potential towards chamber of higher osmotic potential via
semipermeable membrane until equilibrium gets establish. As a result level of liquid
in capillary attached to chamber containing solvent becomes low and vice versa for
other chamber. Difference in height of liquid in capillary helps to determine osmotic
pressure, in return molecular weight also.
Disadvantages: Time consuming and no membrane is perfectly ideal to
completely prevent flow of solute molecules and permit flow of solvent molecules.
(II) Dynamic Membrane Osmometer: Here motion of solvent molecules
towards chamber of solution is stopped by applying equal and opposite pressure on
solution. Thus osmotic pressure is measured in terms that how much pressure has
applied to stop flow of solvent molecules.
Advantages: Equilibrium is established soon between both chambers, so it is
time saving.
(III) High Speed Membrane Osmometers: Here an optical system is used to
monitor flow of solvent molecules through membrane. As soon as solvent molecules
starts penetrate into membrane, optical system automatically adjusts pressure by a
electro-mechanical device to such level so that no solvent molecules will move.
Advantages: Migration of very small polymer molecules across membrane is
perverted, so errors occur in static method do not happen here.
Calculation of Molecular Weight
Van’t Hoff law provides basis of osmometry. For osmotic pressure п, we have
following relation

C

 RT A1  A 2C  A 3C2  A 4C3 .... 
A1, A2, A3 and A4 are virial coefficients, C is concentration of polymer, R is
general gas constant while T is temperature. A 1 equal to 1/ Mn , so above equation
will be
 Polymer Chemistry 851

  1 
 RT   A 2C  A 3C2  A 4C3 .... 
C  Mn 

Fig. 13.18 Functioning of membrane osmometers (a) when both chambers have
solvent, and (b) when one chamber has solvent while other has solution.

Figure 3.12 Plot of ÿ/RTC vs. C used to determine

1/Mn in osmometry. (From Modern Plastics
Technology by R. Seymour, Reston Publishing Fig. 13.19 Plot of п/RTC versus C
Company, Reston, Virginia, 1975)
For very dilute solutions, above equation will be
  1 
 RT   A 2C 
C  Mn 

 1
  A 2C (13.111)
RTC Mn
Following results are drawn from equation (13.111)
(i) Equation (13.98) is a linear equation.

(ii) Plot of п/RTC versus C gives an intercept equal to 1/ Mn and slope equal to A2
(Fig. 13.18). Thus inverse of intercept gives value of Mn .
(iii) Value of A2 is measure of polymer-solvent interactions. High value of slope
indicates good polymer-solvent interactions, so that solvent is considered of
good quality.
852 Modern Physical Chemistry

(iv) Low value of slope gives an indication of poor solvent-polymer interactions.

(v) If value of slope is zero, then graph will be parallel to x-axis, and that solvent
is considered very poor and called theta solvent.
This is how molecular weight is determined by osmometry.
13.8.4 Light Scattering Measurements
If a beam of light is passed through a colloidal solution, it is possible to see
the light beam from sides. This is the well known Tyndall effect which results from
the scattering of a part of the beam of light by the colloidal particles in all directions.
This principle is used here to determine the weight average molecular weight of
polymers.
(a) Light Scattering Measurements from Particles with Diameter Less
than /20
In the case of macromolecules, the colloidal particles are highly solvated and
gel particles consist of about 90 to 99% of the solvent. The refractive index of each
colloidal particle is not very different from that of solvent.
According to P. Debye the following power function applies in such cases
32π3 RTc  ndn  dπ 
Δτ     (13.112)
3λ4 N A  dc  dc 

Where τ is the turbidity, Δτ represents the excess turbidity of the solution

over that of the pure solvent,  is the wavelength of light and n is the refractive
index. In the absence of absorption, τ is related to the primary beam intensity before
and after it passes through the scattering medium. If the incident intensity I o is
reduced to I in a length ℓ of sample, then
I
 e τt
Io

The turbidity which is the total scattering integral over all angles, is often
referred by the Rayleigh ratio R(θ) [ R(θ)  R(θ)  R(θ)solvent ] which relates the scattered
intensity at angle θ to the incident beam intensity. For particles small compared to ,
the Debye equation is obtained as
Kc Hc 1
   2A 2 c  ... (13.113)
R(θ) τ M

Here K is scattering constant and H is Debye factored. Both are related by

following equation
3
K H
16π
2
2π2n2  dn 
and K  
N A λ4  dc 
 Polymer Chemistry 853

2
32π3n2  dn 
H  
3N A λ4  dc 

Equation (13.113) forms the basis of determination of polymer molecular

weight by light scattering. Beyond the measurement of τ or R(θ) , only the refractive
index and the specific refractive index increment (dn/dc) require experimental
determination. The latter quantity is constant for a given polymer, solvent and
temperature, and is measured with an interferometer or differential refractometer.

Equation (13.113) is correct only for vertically polarized incident light and for
optically isotropic particles (in which the refractive index is same in all directions are
said to be isotropic e.g. water, glass). The use of unpolarized light requires that τ
multiplied by (1+cos2θ).

For system with particles size less than /20, any observation angle may be
selected since the scattering function is spherically symmetrical. However for
experimental reasons (to avoid interference of primary beam) one usually selects
values of θ not very different from 90º.

In this way, through the measurements of scattered light intensity at a single

angle, one obtains the molecular weight of particles whose size is less than /20. This
is only true for glycogen, proteins and linear polymers of relatively low molecular
weights solvated in poor solvents.

Fig. 13.21 (A) Essential parts of scattering instrument and (B) scattering of light by
different parts of large molecule

(a) Light Scattering Measurements from Particles with Diameter

Greater than /20

For polymer molecules, whose coil diameter is larger than /20, the scattering
function is no longer spherically symmetrical. Instead one finds a diminution in
intensity of scattered light due to interference, which is different at different
observation angles. This results from the fact that for larger particles, the different
scattering centers within a particle are so far from one another that the resulting
854 Modern Physical Chemistry

scattered rays have a path difference of the order of 0 to /20. Since the separate
scattering centers within a polymer molecule are activated by one and the same
wavelength of primary light, the scattered radiation resulting from the centers is
coherent, and therefore capable of interference. The degree of diminution resulting
from the interference depends upon the path difference which in turn depends on the
angle θ. The scattering of light by larger particles is shown in Fig. 13.21 (b).
Therefore the intensity of scattered light differs depending on the observation angle
θ. The larger is angle θ, the greater is the reduction in scattering intensity R(θ) . The
factor by which the scattering intensity R(θ) (for a perpendicularly polarized incident
beam diminished by interference) is diminished by interference at an angle θ is called
the scattering function P(θ) [where R(θ) equals the scattered light in the immediate
vicinity of the primary beam, i.e. with θ→0]. Therefore, for the scattering intensity
R(θ) reduced to standard conditions and measured at the angle θ, one can write

R(θ)  R(0) P(θ)

For larger molecules, scattering factor must be introduced into Debye

equation (13.113)

Kc 1 1 
   2A 2 c  ...  (13.114)
R(θ) P(θ)  M 

As the macromolecules are prudent in coil form in solutions, so the scattering

factor P(θ) would be dependent upon the scattering angle θ, the wavelength  and the
average coil radius s 2 . The following approximations has been written by Zimm as

1  16π2  2 θ
1 2 
s sin2  
P(θ)  3λ  2

Here s 2 is gyration radius which is the average distance from axis of rotation
at which all the masses could be concentrated to produce the same amount of
moment of inertia that the actual distribution of mass possess.

4π θ
s2  sin
λ 2

Substituting the value of 1/P(θ) into equation (13.114), we get

Kc  1    16π2  2  θ 
   2A 2 c  ...  1   2 
s sin2    (13.115)
R(θ)  M    3λ   2 
 Polymer Chemistry 855

Equation (13.115) has two variables c and θ, thus extrapolations can be made
by varying one and kept the other constant and vice versa (Zimm plot Fig. 13.22).
These extrapolations will help to get useful results.

(I) In the limit of c→0, equation (13.115) becomes

 Kc   1    16π2  2  θ 
     1   2 
s sin2   
 R(θ) c 0  M    3λ   2 

The plot of Kc/ R(θ) at c→0 versus sin2 (θ/2) would yield a straight line with
slope and intercept as

1  16π 2  2
Slope   s
M  3λ2 
1
Intercept 
M

Reciprocal of intercept gives value of molecular weight of polymer.

On dividing the slope with intercept, we get

 Slope  1  16π2  2
    2 
s (M)
 Intercept c 0 M  3λ 

 Slope  3λ2
s2    2
 Intercept c 0 16π

In this way ratio of slope to intercept when c→0 gives value of gyration
radius of polymer coil.

(II) Zimm plot (Fig 13.22) is the representation of light scattering data which can
be used to conduct the following extrapolations of equation (13.115) on a single
graph. In Zimm plot Kc/ R(θ) is plotted against sin2 (θ/2) +kc. k is a constant which is
chosen to give a good display to graph.

 In the limit c→0 and θ→0º, then equation (13.115) becomes

 Kc  1 
    (13.116)
R
 (θ) c 0,θ0o  M

 In the limit c→0 , then equation (13.115) becomes

856 Modern Physical Chemistry

 Kc   1    16π2  2  θ 
     1   2 
s sin2    (13.117)
 R(θ) c 0  M    3λ   2 

 In the limit θ→0º , then equation (13.115) becomes

 Kc  1
    2A 2 c (13.118)
 R(θ) θ0o M

Value of M, A2 and s 2 can be obtained from extrapolated data of equation

(13.116) to (13.118).

Fig. 13.22 Zimm plot in which Kc/ R(θ) is plotted versus sin2 (θ/2) +kc

Table 13.1 Applicable weight range and average molecular weights determined from
different methods
Applicable weight
Method Average
range
End group analysis Mn upto 20,000
Membrane osmometry Mn 20,000 to 20,000,00
Vapour phase osmometry Mn upto 40,000
Cryoscopy Mn upto 40,000
Light scattering Mw upto 50,000
Sedimentation Mz upto 
 3.5 CHROMATOGRAPHY
As will be noted shortly, certain techniques such as colligative methods (Secs. 3.6–3.8),
light scattering photometry, special mass spectral techniques, and ultracentrifugation allow
the calculation of specific or absolute molecular weights. Under certain conditions some
of these allow allow the calculation of the molecular weight distribution (MWD).
These are a wide variety of chromatography techniques including paper and column
techniques. Chromatographic techniques involve passing a solution containing the to-be-
1. tested sample through a medium that shows selective absorption for the different compo-
nents in the solution. Ion exchange chromatography separates molecules on the basis of
their electrical charge. Ion exchange resins are either polyanions or polycations. For a
polycation resin, those particles that are least attracted to the resin will flow more rapidly
through the column and be emitted from the column first. This technique is most useful
for polymers that contain changed moieties.
2. In affinity chromatography, the resin contains molecules that are especially selected
that will interact with the particular polymer(s) under study. Thus, for a particular protein,
the resin may be modified to contain a molecule that interacts with that protein type. The
solution containing the mixture is passed through the column and the modified resin
preferentially associates with the desired protein allowing it to be preferentially removed
from the solution. Later, the protein is washed through the column by addition of a salt
solution and collected for further evaluation.
3. In high-performance liquid chromatography (HPLC), pressure is applied to the col-
umn that causes the solution to rapidly pass through the column allowing procedures to
be completed in a fraction of the time in comparison to regular chromatography.
When an electric field is applied to a solution, polymers containing a charge will
4. move toward either the cathode (positively charged species) or the anode (negatively
charged species). This migration is called electrophoresis. The velocity at which molecules
move is mainly dependent on the electric field and change on the polymer driving the
molecule toward one of the electrodes, and a frictional force dependent on the size and
structure of the macromolecules that opposes the movement. In general, the larger and
more bulky the macromolecule, the greater the resistance to movement, and the greater
the applied field and charge on the molecule the more rapid the movement. While electro-
phoresis can be conducted on solutions it is customary to use a supporting medium of a
paper or gel. For a given system, it is possible to calibrate the rate of flow with the molecular
weight and/or size of the molecule. Here the flow characteristics of the calibration material
must be similar to those of the unknown.
Generally though, electrophoresis is often employed in the separation of complex
molecules such as proteins where the primary factor in the separation is the charge on the
species. Some amino acids such as aspartic acid and glutamic acid contain an “additional”
acid functional group, while amino acids such as lysine, arginine, and histidine contain
“additional” basic groups. The presence of these units will confer to the protein tendencies
to move towards the anode or cathode. The rate of movement is dependent on a number of
factors including the relative abundance and accessability of these acid and base functional
groups.
Figure 3.8 contains an illustration of the basic components of a typical electrophore-
5. sis apparatus. The troughs at either and contain an electrolyte buffer solution. The sample
to be separated is placed in the approximate center of the electrophoresis strip.
Gel permeation chromatography (GPC) is a form of chromatography that is based
on separation by molecular size rather than chemical properties. GPC or size exclusion

Copyright © 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 3.8
Basic components of an electrophoresis apparatus.

Figure. Affinity chromatography

chromatography (SEC) is widely used for molecular weight and MWD determination. In
itself, SEC does not give an absolute molecular weight and must be calibrated against
polymer samples whose molecular weight has been determined by a technique that does
give an absolute molecular weight.
Size exclusion chromatography is an HPLC technique whereby the polymer chains
are separated according to differences in hydrodynamic volume. This separation is made
possible by use of special packing material in the column. The packing material is usually
polymeric porous spheres, often composed of polystyrene crosslinked by addition of vary-
ing amounts of divinylbenzene. Retension in the column is mainly governed by the parti-
tioning (or exchanging) of polymer chains between the mobile (or eluent) phase flowing
through the column and the stagnate liquid phase that is present in the interior of the
packing material.
Through control of the amount of crosslinking, nature of the packing material and
specific processing procedures, spheres of widely varying porosity are available. The
motion in and out of the stationary phase is dependent on a number of factors including
Brownian motion, chain size, and conformation. The latter two are related to the polymer
chain’s hydrodynamic volume—the real, excluded volume occupied by the polymer chain.
Since smaller chains preferentially permeate the gel particles, the largest chains are eluted
first. As noted above, the fractions are separated on the basis of size.
The resulting chromatogram is then a molecular size distribution (MSD). The rela-
tionship between molecular size and molecular weight is dependent on the conformation
of the polymer in solution. As long as the polymer conformation remains constant, which
is generally the case, molecular size increases with increase in molecular weight. The
precise relationship between molecular size and molecular weight is conformation-depen-
dent. For random coils, molecular size as measured by the polymer’s radius of gyration,
R, and molecular weight, M, is proportional to Mb, where b is a constant dependent on
the solvent, polymer concentration, and temperature. Such values are known and appear
in the literature for many polymers, allowing the ready conversion of molecular size data
collected by SEC into molecular weight and MWD.

Copyright © 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 3.9 Sketch showing flow of solution and solvent in gel permeation chromatograph. (With
permission of Waters Associates.)

There is a wide variety of instrumentation ranging from simple manually operated

devices to completely automated systems. Figure 3.9 contains a brief sketch of one system.
Briefly, the polymer-containing solution and solvent alone are introduced into the system
and pumped through separate columns at a specific rate. The differences in refractive
index between the solvent itself and polymer solution are determined using a differential
refractometer. This allows calculation of the amount of polymer present as the solution
passes out of the column.
The unautomated procedure was used first to separate protein oligomers (polypep-
tides) by use of Sephadex gels. Silica gels are also used as the GPC sieves. The efficiency
of these packed columns may be determined by calculating the height in feet equivalent
to a theoretical plate (HETP) which is the reciprocal of the plate count per feet (P). As
shown by the expression in Eq. (3.14), P is directly proportional to the square of the
elution volume (Vc) and inversely proportional to the height of the column in feet and
the square of the baseline (d).
2

p
f d冢 冣
16 Ve
(3.14)

Conversion of retention volume for a given column to molecular weight can be

accomplished using several approaches including peak position, universal calibration,
broad standard and actual molecular weight determination by coupling the SEC to an
instrument that gives absolute molecular weight.

Copyright © 2003 by Marcel Dekker, Inc. All Rights Reserved.

 In the peak position approach, well-characterized narrow fraction samples of known
molecular weight are used to calibrate the column and retention times determined. A plot
of log M vs. retention is made and used for the determination of samples of unknown
molecular weight. Unless properly treated, such molecular weights are subject to error.
The best results are obtained when the structures of the samples used in the calibration
and those of the test polymers are the same.
The universal calibration approach is based on the product of the limiting viscosity
number (LVN) and molecular weight being proportional to the hydrodynamic volume.
Benoit showed that for different polymers elution volume plotted against the log LVN
times molecular weight gave a common line. In one approach molecular weight is deter-
mined by constructing a “universal calibration line” through plotting the product of log
LVN for polymer fractions with narrow MWDs as a function of the retention of these
standard polymer samples for a given column. Molecular weight is then found from reten-
tion time of the polymer sample using the calibration line.
Probably the most accurate approach is to directly connect, or couple, the SEC to
a device, such as a light scattering photometer, that directly measures the molecular weight
for each elution fraction. Here both molecular weight and MWD are accurately determined.

3.6 OSMOMETRY
A measurement of any of the colligative properties of a polymer solution involves a
counting of solute (polymer) molecules in a given amount of solvent and yields a number-
average. The most common colligative property that is conveniently measured for high
polymers is osmotic pressure. This is based on the use of a semipermeable membrane
through which solvent molecules pass freely but through which polymer molecules are
unable to pass. Existing membranes only approximate ideal semipermeability, the chief
limitation being the passage of low molecular weight polymer chains through the mem-
brane.
There is a thermodynamic drive toward dilution of the polymer-containing solution
with a net flow of solvent toward the cell containing the polymer. This results in an increase
in liquid in that cell causing a rise in the liquid level in the corresponding measuring tube.
The rise in liquid level is opposed and balanced by a hydrostatic pressure resulting in a
difference in the liquid levels of the two measuring tubes—the difference is directly related
to the osmotic pressure of the polymer-containing solution. Thus, solvent molecules tend
to pass through a semipermeable membrane to reach a “static” equilibrium, as illustrated
in Fig. 3.10.
Since osmotic pressure is dependent on colligative properties, i.e., the number of
particles present, the measurement of this pressure (osmometry) may be applied to the
determination of the osmotic pressure of solvents vs. polymer solutions. The difference
in height (⌬h) of the liquids in the columns may be converted to osmotic pressure (␲) by
multiplying the gravity (g) and the density of the solution (␳), i.e., ␲  ⌬h␳g.
In an automatic membrane osmometer, such as the one shown in Fig. 3.11, the
unrestricted capillary rise in a dilute solution is measured in accordance with the modified
van’t Hoff equation:
RT
␲ C  BC2 (3.15)
Mn
As shown in Fig. 3.11, the reciprocal of the number average molecular weight
(M1
n ) is the intercept when data for ␲/RTC vs. C are extrapolated to zero concentration.

Copyright © 2003 by Marcel Dekker, Inc. All Rights Reserved.