Size-exclusion Chromatography as a Useful Tool for the...

www.pss-polymer.comIUPAC Workshop, MACRO 2016, Istanbul

Size-exclusion Chromatography as a Useful Tool for the Assessment ofPolymer Quality and Determination of

Macromolecular PropertiesPeter Kilz, PSS Polymer Standards Service, 55023 Mainz, Germany

1. Introduction into Macromolecular Chromatography2. Separation and 2-dimensional Techniques3. Detection and Information Content4. Summary


IntroductionMost polymeric materials are highly complex multi-component materials

even simple polymerization leads to products with multiple property distributions

Analytical Challenges: Determination of distributed properties

physical dispersity (MMD)

chemical dispersity (CCD)

topological dispersity (MAD)

structural dispersity

functional distribution (FTD)

+


Introduction

Characterization Strategy

A) Characterization of bulk materials access to bulk properties / property averages

requires batch methods e.g. Light Scattering (LS), Viscometry, Osmometry, Ultracentrifugation (AUC)NMR, IR, ...

B) Characterization of separated fractions access to property distributions

requires comprehensive chromatography

1) analytical fractionation methods: E.g. Liquid chromatography (SEC, LAC, LCCC) Ultracentrifugation (AUC) Field flow fractionation (FFF) (Gas chromatography: GC)

Mass sprectrometry: MALDI-ToF

2) detection techniques e.g. RI, UV, LS, Viscometry, FTIR, NMR, MS

separation - detection combinations determine which distributions can be measured


Introduction

Chromatographic Modes

a) Size exclusion mode: SEC

KSEC = exp ( ∆S/R)

0 < KSEC < 1 ∆H = 0

b) Adsorption mode: HPLC

KHPLC = exp (- ∆H/RT)

KHPLC > 1 ∆H ā Τ∆S

c) critical adsorption point: LC-CC

K = 1 ∆H = ∆S

log M SEC LC-CC LAC


Chromatographic Modes of Separation

Comparison of Chromatographic Modes

technique separation governed by information content potential problems

SEC • hydrodynamic volume • molar mass (MMD) • calibration dilemma • molecular size in solution • chemical composition (CCD) • specific interactionsdiffusion controlled process

LC-CC • chain inhomogeneity • functionality type (FTD) • irreversible adsorption • defect structures • molecular architecture (MAD) • determination of critical• endgroups adsorption pointdiffusion and adsorptioncontrolled process

HPLC • chemical composition • chemical composition (CCD) • molar mass influence • endgroup • functionality type (FTD) • partial adsorption

• large k'adsorption controlled process


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Introduction

SEC Separation Principles

- solutes diffuse between mobile phase and pores in stationary phase- conformational entropy loss is driving force- retention based on hydrodynamic size in solution Vh- molar mass by rentention calibration or proper detection method

pore volume

Elution volumeV0 Vt

Separation range

I II III


Introduction

SEC Instrumentation

Special instrumental requirements:- solvent compatibility- prevent clogging by solvent evaporation- multi-detector application- columns: mainly polymer packing- often:

absolute concentrations requiredabsolute injection volume required

critical modules:- pumps- autosamplers- software

Degasser

Pump

Injector

Columnthermostat

Detectors

Referencematerials

MCDS


Introduction

SEC Data Systems

Special software requirements:

- long analysis times- complex data treatment- multi-signal processing- determination of distributions- combination of methods- multiple vendor support- integration in existing infrastructure

Signal 1

Signal 2

Signal 3

Signal 4

Methods &Algorithms Results

ResultView 1

ResultView 2

ResultView 3

Plethora of Software Requirements


Introduction

Determination of Property Distributions

complete description of properties and contributions

- accurate determination of amounts- proper measurement/calibration of properties- accurate results calculation and representation

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PSS WinGPC Unity

Amount

Molar mass <----- Increasing molar mass

Elution volume [ml]

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1.4correct molar mass distributionwrong molar mass distribution

Example : Conversion of raw signals to molar mass distribution


Conventional Data Analysis

Determination of fundamental parameters

Chromatogram: relates apparant concentration to elution volume / retention timecalibration curve: relates molar mass to chromatographic positionmolar mass distribution: shows mass fraction of molecules of given molar mass

In all cases:Mn = Mw = Mz

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rel.conc.

position / M

baseline

<----- Increasing molar mass

But...Very differentapplication properties


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Molar Mass [g/mol]

1e 3 1e 4 1e 5 1e 6

Virgin (Mw: 78000 D)

Extruded (Mw: 69000 D)

Recycled (Mw: 59000 D)


Polymer Degradation during Recycling Processes

conditions:

system: PSS SECcurity GPCeluent: TCM/HFIPcolumns: PSS SDV 5µmdetection: UV@260nmsoftware: PSS WinGPCanalysis in: 35 min / sample


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Elution time [min]

analytical: 8x300mm (reference)narrow-bore: 4x250mmPSS HighSpeed: 20x50mm


Conventional and HighSpeed Analysis

Benefits of HighSpeed SEC

- short analysis time (up to 10-fold)- no (additional) sample degradation*- no special SEC hardware required* - no method change*

*) only by using PSS HighSpeed column technology


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Elution time [mim]

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norm

. re

l. co

nc.

[%]


Quality Assurance by HighSpeed SEC

commercial polycarbonate in THFmw by producer: 30000 g/mol

60 repeats in 2h

column: 2x PSS SDV 5 µm HighSpeedcalibration: PSS ReadyCal PS standardsdetection: UV

HighSpeed result:

Mw: (29610±150) g/molRSD: 0.5%



HighSpeed Heparin Quality Assurance

column: PSS HighSpeed Suprema 100, 10 µmanalysis time: 2 mincalibration: Heparin endgroup (DAB); PSS WinGPCdetection: RI

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time [min]

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Heparin AHeparin B

Overlay HighSpeed SEC

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Time

Heparin QC Chart

QC passed

Sample A

Sample B


Chromatographic Modes of Separation

Potential limitations in SEC

• Molecular weight range:Separation range may be increased by using

combination of different pore size columns

Easy to overcome

• Resolution:Separation efficiency may be increased by using

longer or more columns of same pore size

Peak capacity:

Easy to overcome

• Size-separationco-elution of different species (e.g. copolymers, branched molecules)

Can be difficult to meetrequires often differentLC techniques


2-Dimensional Chromatographyn independent properties require n-dimensional methods for accurate (independent) characterization.

Possible multidimensional chromatography techniques:

HPLC, SEC, LC-CC, GC, TREF, GPEC,.....

Example:

combination of LAC(HPLC) and SEC:

1st dimesion:LAC/HPLC for separation according to CC

2nd dimesion:SEC for separation according to MM


SEC results (chromatograms)

1stdimension:HPLCDegasserPump

Autosampler

Column Oven/Column2nd

dimension:SEC

Column SEC

DegasserPump

Transfer Valve

Signals

Detector Waste

Waste

2-Dimensional Chromatography

Investigation of CCD and MMD

Experimental setup:


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Elution Volume [ml]

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UV

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U]

PS

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z #1

main product by-product

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main product by-product

2D ChromatographySample A

SEC Analysis of TPE

sample B failed in the field

- main product looks very similar- similar by-products present

SEC does not track performance differencesSample B

sample A sample BMn [kD] 99 90Mw [kD] 109 103Mw/Mn 1.08 1.14Mp [kD] 108 104by-product 0.8% 1.7%

molar masses by narrow PSt calibration

differences due to composition?


2D ChromatographySample A

Comprehensive 2Dby HPLCxSEC

HPLC tracking compositionSEC tracking molar mass

Sample B2D analysis! corroborates similar MMD! shows similar average PS content! reveals big differences in CCD! contour map shows

- differences easily- 2D property distributions


GPC Elution Volume [ml]

2-Dimensional ChromatographyInvestigation of by-product in motor oil additives

Individual techniques

observations difficult to explain

? ?

LC-CAP Time [min]

? ?



2D results

- main product (region 1)- parallel reaction forms region 2

- two different processes- by-product is homopolymer- by-product has broad MMD

- reaction mixture contains 60% desired product- desired product is narrow in CCD and MMD

Molar Mass [Da]

2D contour map



2D compositional analysis

overlay of 2D separationwith chemical composition

supports - two simultaneous polymerization processes- desired product is copolymer- by-product is homopolymer

Molar Mass [Da]


U K k c Md d Sample Samplei

x= × × ×∑ ( )

Detection Techniques in SEC

Detector Signal Characteristics

Ud: Signal intensity

Kd: Instrument constant

ksample: Sample dependent parameter for spectroscopic detectors: ksample = extinction coefficient, κfor refractive index (RI) detectors: ksample = refractive index increment, dn/dc

note: dependent on solvent composition, T, λcsample: Sample concentration

M: Molar mass

x: Detector dependentfor RI, UV, ELSD: X = 0for on-line LS and MS detectors: X = 1for on-line viscometers: X = Mark Houwink coefficient αfor on-line NMR, osmometers* : X = -1

* not commercially available


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Detector Properties

Detector Signal Characteristics

concentration detectorRefractive index detector (RI)

molar mass sensitive detectorsOn-line light scattering detector

On-line viscosimeter

On-line mass spec (or osmometer)

advanced detector combinations provide comprehensive molecular and structural information



Requirements for Accurate Quantification

RI Detection Corona (ELS) Detection

Linear response (conc-area) Strong non-linear response (even log-log)Stable signal (high repeatability) Poor signal stability (low repeatability)No molar mass influence Molar mass dependentnon-specific detector only non-volatiles detected


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Determination of Chemical Heterogeneity

Multiple Detection in SEC Mode

What we need: c(V), Mc(V) ö xk(M), w(log Mc), Mn,c, Mw,c, DcWhat we have: capp(V), M(V)

capp Madvantages:# uses ordinary SEC equipment# copolymer analysis with same injection# no additional sample preparation

limitations:# statistical copolymers# graft copolymers with high graft density


U1

.

.

Ui

'

f11w1 .. .. ..

.. .. .. ..

.. .. .. ..

.. .. .. fikwk

@ctrue

U1

.

.

Ui

'

f11w1 .. .. ..

.. .. .. ..

.. .. .. ..

.. .. .. fikwk

@ctrue

Determination of Chemical HeterogeneityMultiple Detection in SEC Mode

Approach:

Task 1: derive true c(V) from capp(V)needs multi-detector setup with detector calibration

Ui response in detector ifik response factor for component k in

detector iwk weight fraction of component kctrue concentration of sample

þ absolute concentration of all components k in sample


Determination of Chemical HeterogeneityMultiple Detection in SEC Mode

Determination of copolymer response factors


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resp

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[V]

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frac

tion

w(B

) [%

]

Copolymer, c app

comonomer Acomonomer Bw(B) distribution

Copolymer Analysis of Apparent Chromatogramm

capp ' jk

fdk @ ckcapp ' jk

fdk @ ck



Determination of comonomer concentrations

advantages:# universal approach# no special equipment necessary

limitation:# neighbor-group effects


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Mol

ar m

ass

[Da]

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PS calibration

calculated Mc(V)

PBd calibration

w(V)

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Elution volume [ml]

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%A

18 20



Task 2: Mc(V) from homo polymer calibration, orMc(V) directly from molar mass sensitive detection

lg Mc(V) = Σ wk(V) @ Mk(V)

correct for negligible hetero-contact interactions


Determination of Chemical HeterogeneityInvestigation of ABA block copolymer in SEC Mode

SEC results with PS standards:

Mn 127 kDaMw 353 kDaPD 2.78

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Copolymer Molar Mass [D]

10 4 10 5 10 6 g/mol

UV Signal @260nm

RI Signal

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Bd distribution [%]

Copolymer resultswith multi detection:

Mn 76.3 kDaMw 222 kDaPD 2.91

by PS and PBd calibration



SEC with a light scattering detector: MMD, MAD information

Theoretical Background Light Scattering:

for monodisperse samples, diluted solutions, particle size < λ/20

R(θ) = KAcAM

K: Optical constant, includes refractive index increment (dn/dc)²M: Molar Massc: Concentration

for polydisperse samples with larger partical size (non-isotropic scatterer):

KAc/R(θ) = 1/Mw [1+16/3 π²/λ² <R²>z sin²(θ/2)] + 2 A2Ac


zylindrische Meßzelle

Lichtwellenleiter(Eluat ein/aus: unter/über Ebene)

(Winkelposition)



Theoretical Background Light Scattering:

KAc/R(θ) = 1/Mw [1+16/3 π²/λ² <R²>z sin²(θ/2)] + 2 A2Ac

Light scattering techniques:

LALLS: Low angle laser light scatteringRALLS: Right angle laser light scatteringMALLS: Multi angle laser light scattering

MALLS: PSS SLD7000 detector cell

Sample cell


LS Signal

RI SignalM dn dc

−−

→ ⋅ ( / )2

RILSSEC column



LS can be MALLS, RALLS, LALLS

LS signal: U(LS) = K’ A (dn/dc)2 A c A M

RI singal: U(RI) = K’‘ A c

Molar Mass 1.090.000 130.000 17.800 1.620 DConcentration 0.6334 1.2669 1.2669 1.2669 mg/ml


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Molar mass LS [D]

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Rg = 200 nmpre-peak

Rg = 40 nmshoulder

Rg = 10 nmmain peak

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R(t

het

a) [

1/V

]

Mw

(LS

) [D

]

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PL

AP

TO

P,

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Elution volume [ml]

main peakM = 20 kD

shoulderM = 200 kD

pre-peakM = 20000 kD

Detection Techniques in SECSEC with a light scattering detector: MMD, MAD information

PVB (Poly-vinyl butyral) sample: SEC-MALLS

Results on-line Zimm plot:

molar mass measured for every fraction radius of gyration measured for every fractionMMD MAD



SEC with a viscometer detector: MMD, MAD information

Theoretical Background:

SEC separates according to hydrodynamic volume

Vh,1 = Vh,2

[η]1AM1 = [η]2AM2

A chance to solve the calibration dilemma:Universal calibration curve

M2 = [η]1AM1/[η]2

[η]2 = K A M2α Mark-Houwink equation

Structure information 18 20 22 24 26 28 301

2

3

4

5

6

7

8

PS

PIPPBDPPG

PAMSPMMA

Elution volume [ml]

Universal Calibration of Different Polymers


Visco Signal

RI Signal Sample

−−

→ →[ ]η

RIViscoSEC column


SEC with a viscometer detector: MMD, MAD information

Viscometer signal: U(V) = K’ A [η] A c

RI signal: U(RI) = K’‘ A c

M from universal calibration curve

MMD


Detection Techniques in SECSEC with a viscometer detector: MMD, MAD information

Structure information, MAD:

[η] = K A Mα Mark-Houwink equation

α = 2 rigid rod

1 > α > 0,5 random coil

α = 0,5 random coil,Theta-conditions

α = 0 solid sphere

Branching coefficient g’: g’ = {[η]branched/[η]linear}M



SEC with FTIR detection: CCD, MMD information

Simultaneous separation and identification of fractions


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Elution time [min]

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A

B C D E

F

A: PVC carrier foilB-E: additive packageF: processing agent

on-line FTIR Identification

Mw = 48000 D

Mn = 29500 D

on-line quantification

SEC data processing

Detection Techniques in SECSEC with FTIR detection: CCD, MMD information

type and nature of the polymer used (peak A: PVC)

molar masses and molar mass distributionof the polymer (peak A)

identification of the additives (peaks B - E)

quantification of all additives in the packaging foil

identification and quantification of the processing agent (peak F)


SEC with MS DetectionPotential benefits

• multiple MS techniques: offline: MALDI online: SQ, QQQ, QTOF, ion mobility

• MS measures M with highest accuracy• MS has high resolution• MS can resolve co-eluting species• MS offers very high sensitivity (trace components)• current instruments easy to use


SEC with MALDI-MS DetectionOverview

MALDI advantages:

- absolute molar mass- repeat unit identification- endgroup determination- structure elucidation- high molar mass range

disadvantages:

- matrix influnces- discrimination in polydisperse samples - only offline mode (spotting)- copolymers difficult

GPC-MALDI of PMMARef.: Gores, Pasch; Polymer 36, 1999


SEC with ESI-MS DetectionOverview

online MS advantages:

- absolute molar mass- repeat unit identification- endgroup determination- structure elucidation- identification- high resolution

disadvantages:

- hmw limitations- multiple charges (ESI)- copolymers difficult

Current state of SEC-MS:

- integration in GPC/SEC software- easy-to-use for chromatographers- many automated workflows

m/2e m/e∆m/w 86.09methly acrylate


SEC with ESI-MS DetectionAnalytical Results

- poly(methyl acrylate)- 2 endgroups propyl (43), butyl (57)- simple charge pattern- good mass resolution- 2 main distributions same MA repeat units

SEC-MS reveals: - mixture of species which behave differently in SEC- GPC separation of regular chains- no separation if irregular species

However:still absolute M and MWD


SEC with NMR DetectionBasics

NMR can be used as a (universal) chemical detectorNMR is a chemical sensor looking at local chemical environment

ideal for structure elucidation: chemical shift, J coupling

High-field NMR couling Low-field NMR detection

non-destructive non-destructivesuper-conductive magnet permanent magnethigh resolution low resolutionsmall differences obvious major sample characteristicsexpensive inexpensivecomplex simple to use (detector)large small benchtoptime-consuming low operation costinterfacing difficult flexiblehigh operational cost modular setupsexpert knowledge required saves sample prep time

on same scale


SEC with NMR DetectionHigh-Field NMR Coupling to HPLC

Sample: non-ionic surfacrants

- samples have been stored in storage valve (BPSU)Offline NMR scans

- solvent signals eliminated by NMR pulse sequences

Ref.: Pasch/Hiller (1996), Macromolecules, 2, 6556


SEC with 1H-NMR DetectionCurrent Status

base NMR: Bruker TopSpin, 20 MHz magnetautomatic supression of solvent peaks0.2ml probescan time: 2 secs

run on: PSS SECcurity GPC system, single PSS SDV 5µm column, THF typical SEC injection conditions

Comparison of spectra with different probes

c

b

a

Resolution

500 MHz

20 MHz

20 MHz

20 MHz

0.06ppm (probe 3)

0.11ppm (probe 2)

0.2ppm (probe 1)

[ppm][ppm]

PMMA

PMMA

syndio

PS

1,2-PB

1,4-PB

1,4-PI


Conclusions- comprehensive SEC/GPC is an established and versatile method- plethora of LC and detection methods for structure investigation- information request determines chromatographic strategy- in-depth characterization of MMD, CCD, FTD, MAD, etc. possible- combination of LC modes opens new horizons- increase of peak capacity by 2D chromatography- unbiased investigation of property distributions- mapping of samples or property quantification in 2D- information-rich detectors add identification and

structure elucidation to separation

Size-exclusion Chromatography as a Useful Tool for the...

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