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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
• Passadiluted (!) 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 = Wi / SWi
Ni = Wi / Mi A S ni = 1 i
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 M ? 1 chain with mass 10 n
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 Mn Mw Mz • Final result of SEC: molar Polyquat-a 6.400 9.700 13.900 Polyquat-b 7.500 11.800 17.100 Polyquat-c 8.400 13.200 18.800 mass moments PLUS molar mass distribution!
Overlay Plot: WF / dLog MW Vs. Log Molecular Weigh t Method: pmm aconv-000 4.vcm 1,35 quat-a -2_0 3-02 -200 9_01.vdt : pmm aconv-00 04.vcm quat-b -2_0 3-02 -200 9_01.vdt : pmm aconv-00 04.vcm quat-c-2 _03-02-2 009_01 .vdt : pmmaconv-0004.vcm 1,20 m
c 1,10 v . 4 0 0 0
- 1,00 v n o c a 0,90 Differential distribution (upper m • m p
: 0,80 d o h t
e 0,70 M / t d
v picture) mostly used. . 0,60 1 0 _ 9
0 0,50 0 2 - 2 0
- 0,40 3 0 _ 2 - 0,30 a - t a u
q 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: Cumulative Weight Fraction Vs. Log Molecular Weight Method: pmma conv-0004.vcm 1,00 quat-a-2_03-02-2 009_01 .vdt : pmmaconv-00 04.vcm quat-b-2_03-02-2 009_01 .vdt : pmmaconv-00 04.vcm 0,90 quat-c-2_0 3-02-2009_ 01.vdt : pm maconv-0004.vcm m c v . 0,80 4 0 0 0 - v
n 0,70 o c a m m p
: 0,60 d o h t e M
/ 0,50 t d v . 1 0
_ 0,40 9 0 0 2 - 2 0
- 0,30 3 0 _ 2 - a - t 0,20 a u q
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
0 ≤ K ≤ 1 in which: Vi = Vt –V0 SEC
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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 Page 24
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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 Page • Detection 31
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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=0is 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,Mz1-5%). Very useful for comparative purposes. Page 32
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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 calibration”. Page 33
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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. • Therefore: this method is only limitedly used, nowadays. Page 34
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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 a Mark Houwink relation: [h]= KM . Page 36
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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:
2 2 2 4p h dn 1 16p 2 2 q o æ ö = 1+ < RG > sin ( ) K = 4 ç ÷ 2 Pq 3l 2 Nal è dcø
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. Page 38
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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 >15nm,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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FOR INTERNAL USE ONLY 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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