basics of gpc (sec) separation including calibration options · 9/20/2016 1 for internal use only...
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Basics of GPC (SEC) separationincluding calibration options
Dr. Harry J.A. PhilipsenWorkshop at the International Symposium on GPC/ SEC and related
techniquesAmsterdam, September 26, 2016
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- 2016-now: Senior Scientist/ Project Director and Competence Lead MolecularStructures and Quantification of Synthetic Polymers.
- 2011-2016: Resources manager Polymers Cluster at DSM Resolve, Geleen.
- 2014-now: Project director “(Bio)Macromolecular Characterization” – part ofthe DSM corporate Analysis & Characterization program.
- 2009-2012: Visiting scientist capacity group Polymer Chemistry (SPC) at TU/eand lecturer Analytical Chemistry at TU/e.
- 2007-2010: New Business Development Manager at DSM Resolve, Geleen.
- Until 2007: Researcher/ group leader (analytical chemist) at OcéTechnologies, 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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Some words on myself..
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SEC in industrial applications - 2016
o Big gap between academic research andindustrial practice on polymer separations.
o SEC: one of the most used techniques forpolymer characterization in industry.
o Considered as simple, but still manypitfalls.
o Real life accuracy and precision oftencumbersome..
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GPC and SEC are different names for the same technique;
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• GPC: Gel Permeation Chromatography.
• SEC: Size Exclusion Chromatography: the official IUPAC name.
• Liquid chromatography technique that separates molecules according to theirsize (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 lowmolecular mass molecules of interest).
o MAIN APPLICATION: determination of molar mass averages and molarmass-distribution of polymers/ macro molecules.
What is GPC/ SEC?
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Dispersity of polymers
• Synthetic polymers and somenatural polymers (e.g. starch) arepolydisperse.
• Types of distributionso 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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A polymers’ molar mass and its distribution determine to a large extent final(mechanical) properties.
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• Various methods toassess molar massaverages and molarmass distributions.
• Different statisticalaverages correlate todifferent properties:
o Mn: brittleness.
o Mw: processing.
o Mz: elasticity.
Molar mass distribution
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Various methods to assess molar mass averages and – distributions.
Distributions can ONLY be determined by separation based methods.
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Most applied
Methods for molar mass determination
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• Pass a diluted (!) polymer solutionover a porous gel (packed in acolumn) with a chosen pore size/distribution.
• Pore volume that can be accessedby small molecules is larger thanthat of larger molecules. Smallmolecules are more retained.
• If this process is not influenced byenthalpic (adsorptive) interactionsthen elution volume can becorrelated to molar mass.Therefore: (ΔH = 0) should be met.Else you will end up with mess!
Principle of Size Exclusion Chromatography
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• In essence SEC and HPLC only differin their thermodynamic conditions. InSEC these are chosen such that noenthalpic interactions with thestationary phase occur (ΔH = 0).
• Solvents: in SEC only 1 solvent isused (‘isocratic analysis’); ininteractive forms of HPLC solventprogramming is used (‘gradientelution’).
• Often more than 1 detector is used. InSEC: combination of refractive index(RI), UV (diode array), DifferentialViscometry (DV) and light scattering(LS). In HPLC: combination of UV,Evaporative Light Scattering Detection(ELSD) and MS.
Scheme for SEC (or HPLC)
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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 forretention as the larger molecule (right). Entropy decrease for thelarger molecule is bigger than that of the smaller molecule.
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Separation modes in polymer chromatographyΔG = 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 MassDistribution (MMD).
• Gradient LAC: for ChemicalComposition Distribution(CCD).
• LCCC: for Functional TypeDistribution (FTD), BlockLength Distribution (BLD).
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• Measuring retention of lowpolydispersity polymer standards (Pd<< 1.1) with known molar masses.
• From the obtained calibration curvethe distribution of an unknownpolymer can be transformed in amolar mass distribution with itsstatistical averages.
Calibration of SEC
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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, polyacrylicacid.
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Nomenclature, time sliced peak output
Ai
Number of molecules: Ni
Number fraction: ni = Ni / SNi
Weight of molecules: W i
Weight fraction: wi = W i / SW i
Ni = W i / Mi
S ni = 1S 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 1001 chain with mass 10
Example B:1 chain with mass 10010 chains with mass 10
Mn ?
Mw ?
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Molar mass averages (3)
Example A Example BM1 = mass chain 1 100 100M2 = mass chain 2 10 10x10ni = number fraction 1 ½ 1/11n2 = number fraction 2 ½ 10/11Mn = S niMi 55 18.2w1 = weight fraction 1 = N1M1/SNiMi 10/11 ½w2 = weight fraction 2 = N2M2/SNiMi 1/11 ½Mw = S wiMi 91.8 55z1 = z - fraction 1 = w1M1/SwiMi 100/101 10/11z2 = z - fraction 2 = w2M1/SwiMi 1/101 1/11Mz = 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 beidentical, distribution can differà properties!
• Important to determinedistributions instead of onlyspecific moments.
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Differential versus cumulative mass distribution
• Final result of SEC: molarmass moments PLUS molarmass distribution!
• Differential distribution (upperpicture) mostly used.
Average
Sample Id Mn Mw Mz
Polyquat-a 6.400 9.700 13.900Polyquat-b 7.500 11.800 17.100Polyquat-c 8.400 13.200 18.800
-0,00
1,35
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quat
-a-2
_03-
02-2
009_
01.vd
t/M
etho
d:pm
mac
onv-
0004
.vcm
3,0 4,8L og Molecular Weight
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
Overlay Plot: WF / dLog MW Vs. Log Molecu la r Weigh tMethod : pm m acon v-000 4.vcm
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
0,00
1,00
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quat
-a-2
_03-
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009_
01.vd
t/M
etho
d:pm
mac
onv-
0004
.vcm
3,0 4,8Log Molecular Weight
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
Overlay Plot: Cumulative We igh t Fraction Vs. Log Molecu lar WeightMethod : pm ma conv-0004.vcm
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 quat-c-2_0 3-02-2009_ 01.vdt : pm maconv-0004.vcm
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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 andpore volume
• Limiting value D2 ~ 1/ (3xpore volume)
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Optimizing resolution: column combinations
• For optimizing resolution for aspecific molar mass range ofinterest: we need an appropriatepore 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 Ksecindeed varies between 0and 1. Otherwise: toomuch material elutingaround Ksec 0 or 1.
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How to improve resolution?
• Apply single pore packingwith 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 everySEC column. Strive forlinear calibration curves.Avoid pore mismatch!
• Pore mismatch: unequalvolumes for each poresize range, leading toresolution differences forvarious molar massranges. Distortion ofmolar mass distributions(bending points etc.).
SEC calibration curves of Styragel
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SEC-elution on a column combination ofWaters HR5E, HR4E and HR1. Mind theadditional bending points, distorteddistribution.
Optimizing resolution: pore mismatch
SEC- elution using 2 bimodal PSS-PFGcolumns.
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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 mustbe chosen such that noenthalpic interactions occur:adsorption, association, chargeexclusion!
• Effect is OFTEN overlooked.Using a SEC column doesNOT mean that one is reallydoing SEC.
• In such cases: translation fromelution volume to molar massgoes 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
• Detection
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SEC separates according to hydrodynamic volume, NOT to molar mass. Andonly if the conditionΔH = 0 is met!
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• Hydrodynamic volume of a polymerstrongly depends on its affinity towardsthe solvent (‘solvent quality’).
• Different chemical composition ofstandards and unknown result in relativemolar masses (e.g. ‘polystyreneequivalent molar masses’) that can differup to 100% of the true values.
• Therefore, ‘conventional’ SEC is notaccurate but can be quite precise(reproducibility: Mn 5-10%, Mw, Mz 1-5%).Very useful for comparative purposes.
SEC separates according to hydrodynamic volume
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More on calibration of SEC
For more accurate molar mass values PLUS conformational information: universalcalibration 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. “Universalcalibration”.
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1. K and a of both standards and polymer of interest areknown:
• No additional viscosity detection necessary – Refractive Indexdetection is alone is sufficient.
• K and a values must have been measured under the sameexperimental conditions: solvent and temperature. This is often notthe case.
• Method does not account for k and a being a function of molar mass.
• Therefore: this method is only limitedly used, nowadays.34
Calibration: use of Mark Houwink constants
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2. K and a of both standards and polymer of interest are unknown: adda on-line viscometer to the SEC system in order to determine intrinsicviscosity on-line.
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• Viscometer measures on-linerelative viscosity, ŋrel.
• By combination with theconcentration obtained frome.g. the refractometer, intrinsicviscosity is determined at eachslice by: [ŋ] = (ŋrel / conc.).
• As the hydrodynamic volumeis known at each slice fromcalibration with standards withknown M and measured [ŋ]:Mpolymer can be calculated ateach slide.
Calibration: viscosity detection, universal calibration
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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 afunction of M, leading to concentration errors and therefore also: molarmass 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 bitoverlooked: information on topology differences e.g. branching via theMark Houwink relation: .[ ] ah KM=
Calibration: universal calibration
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• Mark Houwink relation:
o MH ‘a’ (or ‘α’) value is determined by a polymers affinity towards the solvent, itsmolar 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 branchedpolymers.
o Plotting Log vs. Log M (Mark-Houwink plot) providesinformation on topology andsolvent affinity phenomena.
o Changing slope in MH plotmeans changing composition:branching, chemical compositionetc.
Branching information from universal calibration
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• Yet another method for calculating molar masses from SEC: on-linecoupling to light scattering.
o According to light scattering theories, the relation betweenscattered light and molar mass can be described by:
Rθ is the excess Rayleigh scattering ratio of the solution above that of the puresolvent, 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 theinteraction of polymer molecules with each other and which can be ignoredin SEC.
Pθ is the particle scattering factor that can be ignored for small angles.
Calibration: light scattering detection
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o From the combination of on-line light scattering and aconcentration detector, the absolute molar mass can bedetermined at each slice – without calibration.
o For non-isotropic molecules: (Rg > 15 nm, Mw > 100K): detection atvery low angles < 15° needed (RALS) or at multiple angles(MALS). Alternative is combination with viscometry dectection fromwhich a correction for non-isotropic scattering can be made.
o The combination of light scattering and viscosity detection on-lineto SEC is sometimes called “triple SEC” and provides absolutemolar mass and conformational info without calibration.
o Disadvantage of the method: relative insensitive for masses <5000.
Calibration: light scattering detection
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Conventional SEC Universal SEC SLSTarget(DPn)
Mn
(Da)Mw
(Da)PDI Mn
(Da)Mw
(Da)PDI Mw
(Da)FinalProdu
ct(DPn)
30 5700 6200 1.10 n.a. n.a. n.a. 2700 2960 9300 10600 1.14 4800 5600 1.16 5200 5660 9800 11300 1.15 4850 5600 1.17 5400 5790 11300 13900 1.22 5900 7100 1.21 n.a. 69
100 13000 16500 1.27 8700 11000 1.26 9400 102120 13600 18200 1.34 9600 13100 1.38 12100 113
Molecular Weights of block A – Summary
Comparison of molar masses for a poly-oxazoline system as determined byconventional SEC, SEC-DV and off-line static light scattering.
Molar mass determinations: a comparison
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Development of MMD intime during a synthesis,followed by SEC.
Example SEC: following a synthesis
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• Possibilities with SEC for synthetic polymerso 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 scatteringdetection.
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
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• Pitfalls of SEC for synthetic polymerso 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 heavilyunderestimated. Using a SEC column does not automatically meanthat one is doing SEC!
o Accurate, absolute molar mass averages are relatively difficult toassess for relatively low molar mass polymers due to changingrefractive index or UV absorption as function of molar mass (‘end groupeffects’). MS detection is a better alternative for M < 2000.
o Changing composition as function of molar mass (in e.g. radically madecopolymers) cause ‘absolute’ molar masses to be erroneous (changingdn/dc with molar mass).
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SEC – strong and weak points
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