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Basics of GPC (SEC) separation

including calibration options

Dr. Harry J.A. Philipsen

Workshop at the International Symposium on GPC/ SEC and related
techniques
Amsterdam, September 26, 2016

FOR INTERNAL USE ONLY

Some words on myself..

- 2016-now: Senior Scientist/ Project Director and Competence Lead Molecular
Structures and Quantification of Synthetic Polymers.
- 2011-2016: Resources manager Polymers Cluster at DSM Resolve, Geleen.
- 2014-now: Project director “(Bio)Macromolecular Characterization” – part of
the DSM corporate Analysis & Characterization program.
- 2009-2012: Visiting scientist capacity group Polymer Chemistry (SPC) at TU/e
and lecturer Analytical Chemistry at TU/e.
- 2007-2010: New Business Development Manager at DSM Resolve, Geleen.
- Until 2007: Researcher/ group leader (analytical chemist) at Océ
Technologies, Venlo.
-1997-now: Chairman Discussion group Separation methods of Polymers
(DSP) of KNCV.
- 2002-now: Board member and chairman Section Analytical Chemistry
(SAC) of KNCV.
- 1998: PhD Analytical Chemistry (polymer characterization), TU/e.
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SEC in industrial applications - 2016

o Big gap between academic research and

industrial practice on polymer separations.

o SEC: one of the most used techniques for

polymer characterization in industry.

o Considered as simple, but still many

pitfalls.

o Real life accuracy and precision often

cumbersome..

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What is GPC/ SEC?

GPC and SEC are different names for the same technique;

• GPC: Gel Permeation Chromatography.

• SEC: Size Exclusion Chromatography: the official IUPAC name.

• Liquid chromatography technique that separates molecules according to their

size (but only when performed properly).

• Used for:
o Separation and quantification, like in other LC-modes.
o Sample prep (separation of high molecular mass substances from low
molecular mass molecules of interest).
o MAIN APPLICATION: determination of molar mass averages and molar
mass-distribution of polymers/ macro molecules.

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Dispersity of polymers

• Synthetic polymers and some

natural polymers (e.g. starch) are
polydisperse.

• Types of distributions
o Molecular Mass Distribution (MMD).
o Branching distribution.
o Chemical Composition Distribution
(CCD).
o Functional Type Distribution (FTD).
o Charge Density Distribution (CDD).
o Intrinsic Viscosity Distribution (IVD).

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Molar mass distribution

A polymers’ molar mass and its distribution determine to a large extent final
(mechanical) properties.

• Various methods to
assess molar mass
averages and molar
mass distributions.

• Different statistical
averages correlate to
different properties:
o Mn: brittleness.
o Mw: processing.
o Mz: elasticity.

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Methods for molar mass determination

Various methods to assess molar mass averages and – distributions.
Distributions can ONLY be determined by separation based methods.

Most applied

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Principle of Size Exclusion Chromatography

• Pass a diluted (!) polymer solution
over a porous gel (packed in a
column) with a chosen pore size/
distribution.
• Pore volume that can be accessed
by small molecules is larger than
that of larger molecules. Small
molecules are more retained.
• If this process is not influenced by
enthalpic (adsorptive) interactions
then elution volume can be
correlated to molar mass.
Therefore: (ΔH = 0) should be met.
Else you will end up with mess!

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Scheme for SEC (or HPLC)

• In essence SEC and HPLC only differ
in their thermodynamic conditions. In
SEC these are chosen such that no
enthalpic interactions with the
stationary phase occur (ΔH = 0).
• Solvents: in SEC only 1 solvent is
used (‘isocratic analysis’); in
interactive forms of HPLC solvent
programming is used (‘gradient
elution’).
• Often more than 1 detector is used. In
SEC: combination of refractive index
(RI), UV (diode array), Differential
Viscometry (DV) and light scattering
(LS). In HPLC: combination of UV,
Evaporative Light Scattering Detection
(ELSD) and MS.
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Retention in chromatography

Distribution coefficient: K = cs/cm

K = as/am exp(– ΔG/RT)

ΔG = ΔH –T ΔS

Retention factor: k' = ns/nm = (cs.Vs) / (cm.Vm)

k' = K (Vs/ Vm)

k' = (tr - t0) / t0

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Entropy of macromolecular retention in a pore

The smaller molecule (left) has 4 times as many possibilities for

retention as the larger molecule (right). Entropy decrease for the
larger molecule is bigger than that of the smaller molecule.

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Separation modes in polymer chromatography

ΔG = ΔH –T ΔS

SEC: ΔH = 0 → ΔG = TΔS

KSEC = exp(ΔS/R) 0 ≤ KSEC ≤ 1

LAC: TΔS << ΔH → ΔG ≈ ΔH

KLAC = exp(– ΔH/RT) KLAC ³ 1

LCCC: ΔG≈0

LAC: Liquid Adsorption Chromatography

LCCC: Liquid Chromatography under Critical Conditions
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Chromatography of polymers

• SEC: for Molecular Mass

Distribution (MMD).

• Gradient LAC: for Chemical

Composition Distribution
(CCD).

• LCCC: for Functional Type

Distribution (FTD), Block
Length Distribution (BLD).

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Calibration of SEC

• Measuring retention of low

polydispersity polymer standards (Pd
<< 1.1) with known molar masses.

• From the obtained calibration curve

the distribution of an unknown
polymer can be transformed in a
molar mass distribution with its
statistical averages.

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Narrow standards (Refractive Index-signals)

Commercially available:

Mw/Mn <1.1:
polystyrene, PMMA, PEO.

Mw/Mn <1.2: pullulan.

Mw/Mn <1.3: polyethylene.

Mw/Mn >1.3:
polydextran, polyacrylic
acid.

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Nomenclature, time sliced peak output

Number of molecules: Ni
Number fraction: ni = Ni / SNi
Weight of molecules: Wi
Weight fraction: wi = W i / SW i

Ni = W i / Mi
S ni = 1 Ai

S wi = 1

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Molar mass averages (1)

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Molar mass averages (2)

Molar mass averages according to number or mass:

Mn = S niMi Mw = S wiMi

Example A:
1 chain with mass 100
Mn ?
1 chain with mass 10

Example B:
1 chain with mass 100
Mw ?
10 chains with mass 10

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Molar mass averages (3)

Example A Example B
M1 = mass chain 1 100 100
M2 = mass chain 2 10 10x10
ni = number fraction 1 ½ 1/11
n2 = number fraction 2 ½ 10/11
Mn = S niMi 55 18.2
w1 = weight fraction 1 = N1M1/SNiMi 10/11 ½
w2 = weight fraction 2 = N2M2/SNiMi 1/11 ½
Mw = S wiMi 91.8 55
z1 = z - fraction 1 = w1M1/SwiMi 100/101 10/11
z2 = z - fraction 2 = w2M1/SwiMi 1/101 1/11
Mz = S ziMi 99.1 91.8

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MMD moments

D= Mw/Mn
Mn : Impact strength

Mw : Melt viscosity

Mz : Elastic properties of the melt

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MMD’s with identical moments

• Specific moments may be

identical, distribution can differ
à properties!

• Important to determine
distributions instead of only
specific moments.

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Differential versus cumulative mass distribution

Average

Sample Id

Polyquat-a
Mn

6.400
Mw

9.700
Mz

13.900
• Final result of SEC: molar
Polyquat-b
Polyquat-c
7.500
8.400
11.800
13.200
17.100
18.800 mass moments PLUS molar
mass distribution!
Overlay Plot: WF / dLog MW Vs. Log Molecu la r Weigh t
Method : pm m acon v-000 4.vcm
1,35 quat-a -2_0 3-02 -200 9_01.vdt : p mm aconv-00 04.vcm
quat-b -2_0 3-02 -200 9_01.vdt : p mm aconv-00 04.vcm
quat-c-2 _03-02-2 009_01 .vd t : pm maconv-0004.vcm
1,20
quat-a-2_03-02-2009_01.vdt / Method: pmmaconv-0004.vcm

•
1,10

1,00

0,90 Differential distribution (upper

0,80

0,70

0,60
picture) mostly used.
0,50

0,40

0,30

0,20

0,10

-0,00

3,0 3,1 3,2 3 ,3 3,4 3,5 3,6 3,7 3,8 3,9 4,0 4,1 4 ,2 4,3 4,4 4,5 4,6 4,7 4,8
L og Molecular Weight

Overlay Plot: C umulative We igh t Fraction Vs. Log Molecu lar Weight
Method : pm ma conv-0004.vcm
1,00 quat-a-2_03-02-2 009_01 .vdt : pmm aconv-00 04.vcm
quat-b-2_03-02-2 009_01 .vdt : pmm aconv-00 04.vcm
0,90 quat-c-2_0 3-02-2009_ 01.vdt : pm maconv-0004.vcm
quat-a-2_03-02-2009_01.vdt / Method: pmmaconv-0004.vcm

0,80

0,70

0,60

0,50

0,40

0,30

0,20

0,10

0,00

3,0 3,1 3 ,2 3,3 3,4 3,5 3,6 3,7 3 ,8 3,9 4,0 4 ,1 4,2 4,3 4,4 4,5 4,6 4,7 4,8
Log Molecular Weight

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Optimizing resolution for a wider molar mass range

Ksec = (VR – V0) / Vt – V0 → VR= V0 + Ksec Vi

in which: Vi = Vt – V0 0 ≤ KSEC ≤ 1

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Resolution concept in SEC of Polymers

Rsp = 0.58/sD2

• Rsp: Resolution in SEC

• D2: slope of calibration curve –

determined by pore size distribution and
pore volume

• Limiting value D2 ~ 1/ (3xpore volume)

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Optimizing resolution: column combinations

• For optimizing resolution for a

specific molar mass range of
interest: we need an appropriate
pore size (combination).
o Single pore (approx. 1.6
decades).
o Bank of individual pore-sizes.
o Mixed bed columns”.
o Bimodal concept.

Calibration curves of various LiChrospher columns

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Optimizing resolution: column selection

Take care that Ksec

indeed varies between 0
and 1. Otherwise: too
much material eluting
around Ksec 0 or 1.

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How to improve resolution?

• Apply single pore packing

with appropriate range.
• Increase plate number by:
o Decreasing flow-rate.
o Increasing temperature.
o Use of smaller particle
size of the packing.
o More columns with the
same PSD.

Effect of psd (a,b) and pore volume (b,c,d).

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Optimizing resolution: pore mismatch

• DO NOT combine every

SEC column. Strive for
linear calibration curves.
Avoid pore mismatch!

• Pore mismatch: unequal

volumes for each pore
size range, leading to
resolution differences for
various molar mass
ranges. Distortion of
molar mass distributions
(bending points etc.).

SEC calibration curves of Styragel

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Optimizing resolution: pore mismatch

SEC-elution on a column combination of

Waters HR5E, HR4E and HR1. Mind the
additional bending points, distorted
distribution.

SEC- elution using 2 bimodal PSS-PFG

columns.

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SEC calibration curves of linear columns

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For proper SEC, condition: ΔH must be met!!

• Combination of column, eluent +

additives and temperature must
be chosen such that no
enthalpic interactions occur:
adsorption, association, charge
exclusion!

• Effect is OFTEN overlooked.

Using a SEC column does
NOT mean that one is really
doing SEC.

• In such cases: translation from

elution volume to molar mass
goes wrong!
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Proper SEC means: many critical parameters

• Solvent selection
• Column selection:
o Particle size and size distribution
o Quality of the packing

• Flow-rate
• Temperature
• Extra column contribution (capillaries, detectors).
• Injection volume
• Injected mass
• Sample:
o Solution viscosity sampling
o Sample preparation
o Sample concentration
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SEC separates according to hydrodynamic volume

SEC separates according to hydrodynamic volume, NOT to molar mass. And
only if the condition ΔH = 0 is met!

• Hydrodynamic volume of a polymer

strongly depends on its affinity towards
the solvent (‘solvent quality’).

• Different chemical composition of

standards and unknown result in relative
molar masses (e.g. ‘polystyrene
equivalent molar masses’) that can differ
up to 100% of the true values.

• Therefore, ‘conventional’ SEC is not

accurate but can be quite precise
(reproducibility: Mn 5-10%, Mw, Mz 1-5%).
Very useful for comparative purposes.
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More on calibration of SEC

For more accurate molar mass values PLUS conformational information: universal
calibration via on-line measuring of intrinsic viscosities (viscosity detection).

Two approaches:
1. K and a of both standards and polymer of interest are known: calculate M.
2. Only molar mass of standards is known: measure [ŋ], calculate M. “Universal
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calibration”.
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Calibration: use of Mark Houwink constants

1. K and a of both standards and polymer of interest are
known:

• No additional viscosity detection necessary – Refractive Index

detection is alone is sufficient.
• K and a values must have been measured under the same
experimental conditions: solvent and temperature. This is often not
the case.
• Method does not account for k and a being a function of molar mass.

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• Therefore: this method is only limitedly used, nowadays.
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Calibration: viscosity detection, universal calibration

2. K and a of both standards and polymer of interest are unknown: add
a on-line viscometer to the SEC system in order to determine intrinsic
viscosity on-line. • Viscometer measures on-line
relative viscosity, ŋrel.

• By combination with the

concentration obtained from
e.g. the refractometer, intrinsic
viscosity is determined at each
slice by: [ŋ] = (ŋrel / conc.).

• As the hydrodynamic volume

is known at each slice from
calibration with standards with
known M and measured [ŋ]:
Mpolymer can be calculated at
each slide.

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Calibration: universal calibration

Universal calibration compared to conventional calibration:

• Provides more accurate molar mass averages, especially from M >
(approximately) 2000. Still: errors up to 10-20% not uncommon.
• For masses < 2000: refractive index and/ or UV absorption are a
function of M, leading to concentration errors and therefore also: molar
mass errors in universal calibration.
• Universal Calibration is more complex than conventional SEC:
o Polymer concentrations must be known accurately.
o Even more strict control of all experimental variables.
o Method strongly influenced by Enthalpic effects.
o Influence of conformational differences.
• Additional, strong element of universal calibration which is often a bit
overlooked: information on topology differences e.g. branching via the
Mark Houwink relation: [h ] = KM
a
.
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Branching information from universal calibration

• Mark Houwink relation:
o MH ‘a’ (or ‘α’) value is determined by a polymers affinity towards the solvent, its
molar mass and its topology.
o The higher the affinity the higher a. Random coil polymers: a ≈ 0.7, rigid polymers: a
> 0.8.
o Low molar mass polymers (M < 5000): a decreases towards 0.5.
o Branched, more compact polymers: a decreases towards < 0.2 for hyper branched
polymers.

o Plotting Log vs. Log M (Mark-

Houwink plot) provides
information on topology and
solvent affinity phenomena.
o Changing slope in MH plot
means changing composition:
branching, chemical composition
etc.

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Calibration: light scattering detection

• Yet another method for calculating molar masses from SEC: on-line
coupling to light scattering.
o According to light scattering theories, the relation between
scattered light and molar mass can be described by:

4p 2ho2 æ dn ö
2
1 16p q
K= ç ÷ = 1 + 2 < RG 2 > sin 2 ( )
3l
Nal4 è dc ø Pq 2

Rθ is the excess Rayleigh scattering ratio of the solution above that of the pure
solvent, measured at angle θ with respect to the incident beam.
M is the molecular weight of the polymer sample.
C is the sample concentration.
A2 is the second virial coefficient of the solution, which corrects for the
interaction of polymer molecules with each other and which can be ignored
in SEC.
Pθ is the particle scattering factor that can be ignored for small angles.
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Calibration: light scattering detection

o From the combination of on-line light scattering and a
concentration detector, the absolute molar mass can be
determined at each slice – without calibration.

o For non-isotropic molecules: (Rg > 15 nm, Mw > 100K): detection at

very low angles < 15° needed (RALS) or at multiple angles
(MALS). Alternative is combination with viscometry dectection from
which a correction for non-isotropic scattering can be made.

o The combination of light scattering and viscosity detection on-line

to SEC is sometimes called “triple SEC” and provides absolute
molar mass and conformational info without calibration.

o Disadvantage of the method: relative insensitive for masses <

5000.
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Molecular Weights of block A – Summary

Molar mass determinations: a comparison

Conventional SEC Universal SEC SLS

Target Mn Mw PDI Mn Mw PDI Mw Final
(DPn) (Da) (Da) (Da) (Da) (Da) Produ
ct
(DPn)
30 5700 6200 1.10 n.a. n.a. n.a. 2700 29
60 9300 10600 1.14 4800 5600 1.16 5200 56
60 9800 11300 1.15 4850 5600 1.17 5400 57
90 11300 13900 1.22 5900 7100 1.21 n.a. 69
100 13000 16500 1.27 8700 11000 1.26 9400 102
120 13600 18200 1.34 9600 13100 1.38 12100 113

Comparison of molar masses for a poly-oxazoline system as determined by

conventional SEC, SEC-DV and off-line static light scattering.

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Example SEC: following a synthesis

Development of MMD in
time during a synthesis,
followed by SEC.

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SEC – strong and weak points

• Possibilities with SEC for synthetic polymers

o Easy and precise (RSD 2-5%): relative molar mass distributions of
samples series of the same polymer type.
o Information of chemical composition variations as function of molar
mass (UV/ RI) that may be indicative for mixtures or composition drift.
o A more accurate but less precise (RSD ≈ 10%) approximation of true
molar mass averages via viscosity detection and light scattering
detection.
o Information on topology- e.g. branching via Mark Houwink plots.

SEC can provide a wealth of information on polymer composition(-

differences).

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SEC – strong and weak points

• Pitfalls of SEC for synthetic polymers

o Results are critically dependent on quite a number of experimental
variables. In practice this is often underestimated.
o Non-exclusion effects leading to bad reproducibility and wrong
‘absolute’ values from e.g. viscosity detection. This problem is heavily
underestimated. Using a SEC column does not automatically mean
that one is doing SEC!
o Accurate, absolute molar mass averages are relatively difficult to
assess for relatively low molar mass polymers due to changing
refractive index or UV absorption as function of molar mass (‘end group
effects’). MS detection is a better alternative for M < 2000.
o Changing composition as function of molar mass (in e.g. radically made
copolymers) cause ‘absolute’ molar masses to be erroneous (changing
dn/dc with molar mass).

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