Foundation GPC Part 2 – Basic
Gel Permeation Chromatography
Introduction
 This presentation introduces gel permeation chromatography (GPC)
 The mechanism of GPC shall be discussed
 The method of analysing data shall be introduced
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Types of Liquid Chromatography
Interactive adsorption, partition, ion exchange, etc
Non-interactive GPC, SEC, GFC
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Nomenclature
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Gel Permeation Chromatography
GPC
Size Exclusion Chromatography
SEC
Gel Filtration Chromatography
GFC
What is a GPC System?
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1.
Reciprocating piston pump delivers eluent from
reservoir at a constant volumetric flow rate.
GPC is always an isocratic separation
2.
Two
position
injection
valve
permits
introduction of sample solution without
interrupting solvent flow. GPC tends to use
larger injection volumes than HPLC (typically
up to 200ul)
3.
GPC columns perform a separation based on
the molecular size of polymer molecules in
solution. Resolution and/or resolving range is
increased by use of multiple column systems
4.
Detector responds to concentration of polymer
molecules eluting from the GPC columns
Additional Components Used in GPC
Concentration detectors
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Differential refractometer (RI)
Ultraviolet absorbance (UV)
Evaporative light scattering or mass detector (ELS, EMD)
Infra-red (IR)
Molecular weight sensitive detectors
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Viscometry
Light scattering
Additional systems
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 Online degasser
 Autosampler
 Column oven
 Additional specific detectors
Polymer Molecules in Solution
 GPC is based on the behaviour of polymer molecules in solution
 In the solid state polymers can be considered like spaghetti – a confusing
mass of intertwined chains
 In solution, polymer molecules are discrete entities
 Due to entrophic effects all but the most rigid of polymer chains curls up in
solution to form a ball like shape
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GPC Column Packings
 GPC
columns are packing with cross-linked, insoluble beads, typically copolymers of styrene and divinyl benzene for organic GPC
 These beads have a rigid pore structure that remains intact in the presence
of solvent
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Synthesis of Porous Beads
 High
cross-link content
gives a rigid, low swelling
product with a welldefined pore structure
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Permeation of Polymer Molecules
 Polymer
coils in solution can
permeate the pores on GPC
packing materials
 Exclusion,
partial permeation
and total permeation are possible
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GPC Separation Mechanism
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Polymer is prepared as a dilute solution in the
eluent and injected into the system
The GPC column is packed with porous
beads of controlled porosity and particle size
Large molecules are not able to permeate all
of the pores and have a shorter residence
time in the column
Small molecules permeate deep into the
porous matrix and have a long residence time
in the column
Polymer molecules are separated according
to molecular size, eluting largest first,
smallest last
Elution Profiles
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As a result of the GPC separation
mechanism, polymer molecules elute
from the column in order of size in
solution
Largest elute first, smallest elute last
The separation is purely a physical
partitioning, there is no interaction or
binding
The separation is isocratic
If polymer molecules have the same
molecular dimensions, they will co-elute
by GPC and may not be separated by
this technique
The calibration curve describes how
different size molecules elute from the
column
GPC Column Technology
 Columns are packed with porous particles, controlled pore size and particle
size
 Columns are produced by slurry packing technique, packed at pressures in
excess of 2000psi
 Column dimensions typically 7-8mm i.d., 250-600mm in length
 Exclusion volume (Vo) - Upper MW limit (also known as void volume)
 Total permeation volume (Vt) – Lower MW limit
 Pore volume (Vp) – Working resolving range of MW
Vp = Vt - Vo
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Typical Calibration Curves for PLgel
Individual Pore Size Columns
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Determination of Polymer Molecular
Weight Distribution by GPC
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Produce a GPC calibration curve for
the column set relating log M to
retention time (RT)
Chromatograph the polymer sample
Normalise and integrate the GPC
response versus retention time plot
for the polymer sample
Convert retention time to logM via
the GPC calibration curve
Present a logM distribution plot and
calculate molecular weight averages
(Mn, Mw) for the distribution
Peak Integration in GPC
The sample peak when integrated can be assumed to be a histogram
consisting of a number of individual “slices”
For each slice, i, the
molecular weight (Mi)
can be derived from the
RT and the number of
molecules (Ni) from the
detector response
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Calibration of GPC Columns
Using Narrow Standards
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Chromatograph a series of well
characterised, narrow polydispersity
polymer standards
Plot peak retention time (RT) versus
peak log molecular weight (logM)
Fit the data using a mathematical
function
The calibration curve will be
characteristic of the GPC column
set used
Performing Narrow Standard GPC
Calibration
Injections of multiple narrow standards reduces the
time taken to calibrate the system
Injection 1
Injection 2
Standards peaks in each
chromatogram must be
fully resolved to obtain
repeatable retention times
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EasiCal Pre-prepared Calibrants
Spatula B
Spatula A
EasiCal PS-1 separation on
3 x PLgel 10µm MIXED-B
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Narrow Standards Available from PL
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Polymer Calibrants for GPC
Mn
- number average molecular weight
Mw
- weight average molecular weight
Mv
- viscosity average molecular weight
Mp
- peak molecular weight
Mw/Mn
- polydispersity by GPC
Must be extremely well characterised
Most commonly used polymer calibrants
Polystyrene - THF, toluene, chloroform, TCB
Polymethyl methacrylate - MEK, ethyl acetate, acetone, DMF
Polyethylene oxide/glycol - aqueous eluents, DMF, DMSO
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Example Certificates of Analysis
Individual standard
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EasiVial
Calibration Methods for Conventional GPC
Aim : to produce a
mathematical model for log
M versus retention time
Narrow standards
Broad standards
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Hamielec
Broad on Narrow
Integral
Curve Fitting for Narrow
Standards Calibration
Polynomial
All data points fitted with one function of the form
Log M = A + B(t)
Linear (1st order)
Log M = A + B(t) + C(t2)
Quadratic (2nd order)
Log M = A + B(t) + C(t2) + D(t3)
Cubic (3rd order)
Cubic spline
Sets of points fitted with a series of cubic equations
Point to point
Points fitted with a series of linear equations
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Calibration of GPC Columns
Using Narrow Standards
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Chromatograph
a series of
well
characterised,
narrow
polydispersity
polymer standards
Plot peak retention time (RT) versus peak
log molecular weight (logM)
Fit the data using a mathematical function
(e.g. polynomial order 1,2,3, etc)
The calibration curve will be characteristic
of the GPC column set used
Errors Due to Limited Calibration Region
The column calibration
should cover the full
elution time region of
the sample to avoid
errors
due
to
extrapolation
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Broad Standard Calibration Hamielec Method
 Requires a single polydisperse sample where Mn and Mw are known
 Assumes a linear calibration Log M = A + B(t)
 Requires only a single injection to calibrate
1. Chromatograph broad standard
2. Input actual Mn and Mw
3. Computer searches for suitable values of A and B that when back
calculated give the correct values for Mn and Mw
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Broad Standard Calibration Broad on Narrow Method
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Requires a series of narrow standards plus a broad
standard of known Mn and Mw
Valid for non-linear calibration curves
Requires several injections to calibrate
1. Chromatograph the narrow standards and fit the data with a
mathematical function log Mns = F(t)
2. Input the Mn and the Mw values for the broad standard
3. Chromatograph the broad standard
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Broad Standard Calibration Broad on Narrow Method
Universal Calibration theory predicts that
log Mbs
=
log (Kns/Kbs) +
(1+abs)
(1+ans) log Mns
(1+abs)
As Kns, Kbs, ans and abs are constants this can be simplified to
log Mbs = A log Mns + B
The computer searches for suitable values of A and B that results in the
correct values of Mn and Mw for the broad standards when back calculated.
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Broad Standard Calibration Integral Method
Requires a broad standard which has been fully characterised to give a
cumulative molecular weight table, e.g.
Log M
2.800
2.865
2.929
…..
5.705
5.789
5.870
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Wt %
0.000
0.005
0.020
99.80
99.99
100.00
Accuracy is improved
with increased number
of data points, usually
50-60 points are used
There are few broad
standards
available
with this level of
characterisation
Broad Standard Calibration Integral Method
The
broad
standard
is
chromatographed and integrated,
the log M values are assigned to
the distribution based on the wt %
values quoted in the table. This
results in a calibration table with
as many points as there are
entries in the table.
The Log M versus retention time
data is then fitted with a
mathematical function as usual.
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Use of Mark-Houwink Constants in GPC
There are few well characterised polymer standards for GPC calibration.
Therefore “unknown” polymer molecular weight can be derived from a GPC
calibration curve obtained using polymer standards and applying the following
theory:
 Molecular size = hydrodynamic volume (HV)
 HV = M [n], where M is molecular weight and [n] is intrinsic viscosity
 In GPC molecules with the same HV elute at the same retention time
 Therefore for different polymer types
M1 [n]1=M2 [n]2
 For a given polymer system Mark-Houwink equation applies : [n] = K M 
 Rearranging these relationships
logM2 =
log(K1/K2)
+
(1+ 1) logM1
(1+ 2)
(1+ 2)
where K1, 1 for polymer standards and K2,  2 for polymer under
investigation are known
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Interpreting Chromatograms
 The
data obtained in a GPC experiment will be in the form of a
chromatogram showing detector response as a function of retention time
 There are fundamental parameters that are present on all chromatograms
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Peak Separation
 Peak separation in GPC is dependent upon resolution and on molecular size
 If two samples have different molecular sizes, then they will be separated to
baseline assuming there is sufficient resolution
 However,
if samples are the same molecular size, then they cannot be
separated by GPC as the mechanism of SEC is based upon size
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 If two peaks are not baseline resolved in GPC, they cannot be analysed as
two separate peaks
 Although
the calculations will give you results, the numbers will be
meaningless as not all of the peak is defined and so the distribution will not be
correct
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Excluded Peaks
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Partial Exclusion
 The
dead space of the separation will be around half of the total elution
volume
 Peaks eluting close to this volume may be partially excluded
 Look for sharp peaks at the front of your chromatograms
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Oligomeric Resolution
 In
GPC, the relationship
between molecular weight
and retention time is
logarithmic
 As
a result,
peaks
equidistant in molecular
weight elute closer together
with increasing molecular
weight
 This
is a classic way to
tell a separation is based on
SEC
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 With
some columns it is
possible to calibrate the
column using the oligomers
 The molecular weights of
the initiator fragment and
the repeat unit of the
polymer must be known
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Interpreting Molecular Weight Distributions
 The
molecular weight
distribution
shows
the
amount of material present
as a function of the
molecular weight
 The MWD looks a bit like
a ‘mirror image’ of
chromatogram
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the
Effect of Baseline Position
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 The
whole peak should
be analysed to get a true
reflection of the sample
 The peak should go down
to the baseline on either
side
 Leaving
out components
of the peak will leave an
‘incomplete’ MWD
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