Interaction Polymer Chromatography

INTERACTION POLYMER CHROMATOGRAPHY

Yefim Brun*, Chris Rasmussen, B. McCauley

DuPont Central Research & Development [email protected]

GPC/SEC and Related Techniques, Frankfurt, 2014

Liquid chromatography for polymer characterization 2

Dissolution Separation Detection Data Reduction

Separation media

• porous particles (HPLC, UPLC)

• non-porous particles (HDC) • monolithic columns • capillary

Size Exclusion Chromatography Interaction Polymer Chromatography SEC IPC

separation by molecular size separation by everything but size

2D-Chromatography IPC/SEC, IPC/IPC, SEC/IPC Molecular properties of polymers & other nanostructures 3

Molar mass, End-groups Topology size and shape star telechelic comb linear Homopolymers

Chemical Composition and Microstructure

statistical alternating block gradient Copolymers

Size, shape, interaction, surface chemistry Biomolecules, nanoparticles, micelles, vesicles

Molecular heterogeneities (distributions): strong effect on end-use properties

Synthesis Molecular Structure Property DuPont vs. polymer characterization community 4

Open literature DuPont

1. SEC is by far the most popular 1. IPC/SEC: 50/50(%) LC technique for polymer characterization

2. IPC: mostly isocratic 2. IPC: gradient approaches separations (LCCC+ barrier (including TGIC) >90% techniques)

3. HPLC practitioner: do not 3. IPC: practically all separations use isocratic mode in problems are more effectively separation of big molecules! solved in gradient mode (vs. isocratic)

Perception: more difficult to control the mechanism of separation in gradient mode How to control separation 5

• Order of elution (selectivity of separation) is determined by mechanism of retention

• Retention is result of interaction between molecules and solid surface of porous particles • Right balance between different types of interaction is the key to separation mechanism Steric Interaction (SEC)

Critical point of adsorption (LCCC) IPC

Adsorption Phase Transition / Partition (HPLC) (Precipitation LC)

LCCC: LC at critical conditions, or LC at critical point of adsorption (CPA)

Enthalpy gain Entropy loss

Mutual compensation effect : MW-independent elution Different modes of isocratic separation 6

® Elution of Narrow Polystyrenes on Symmetry C18

THF Mobile phase THF- ACN (45/55) 474 5,570

474

710,000

5,570

ti tliq 9,100 18,100

2.6 3.0 3.4 3.8 4.2 4.6 5.0 4 6 8 10 12

Elution time tR, min (flow rate 0.5ml/min) THF suppresses non-polar interaction Non-polar interaction prevails

SEC mode at Φ =100%THF : big elutes first Adsorption mode at Φ = 45%THF : big elutes last (or does not elute at all)

Different modes of isocratic separation 7

® Elution of Narrow Polystyrenes at Symmetry C18

Critical Point of Adsorption (CPA) Elution at different compos. Φ (THF,%) 6.0 THF-ACN (48/52) 474 SEC LCCC LAC

5.5 50

Log M Log 5.0 100 Φ = 48% 37,900 CR 45 4.5 80 40 4.0

3.5 186,000 70 t liq 3.0 V 0 Vliq

3 4 5 6 7 8 9 10 11 12 13 0 1 2 3 4 5 6 tR, min (flow rate 0.25ml/min) Elution volume VR (mL)

Transition mode: MW-independent elution at Φ = Φcr (48%THF)

LCCC 8

Applications Limitations

•Separation by functional groups •Often limited to oligomers/low-M polymer separations in wide-pore columns •Separation of block-copolymers by length of individual blocks •Very sensitive to experimental conditions •Separation of homopolymer blends •Temperature/solvent composition fluctuations resulting in peak splitting, • Not applicable to statistical copolymers limited mass recovery

LCCC of 186K at different flow rates Flow rate (ml/min): Nova-Pak C18 Column (60A, 4 µkm, 3.9 x 300 mm) THF-ACN (49/51) 0.5 0.25 0.125

0 5 10 15 20 25 Minutes 30 Let’s discuss after Falkenhagen’s and Hiller’s talks LCCC: separation by end groups 9

Elution of linear PEG (MW=40K) with different end groups at CPA on Symmetry® C4

Critical point of adsorption (CPA): 50% ACN in H2O on Symmetry® C4

Δt = 0.13 mono-brominated PEG KLCCC = 1 + q PEG standard with 2 OH ends

Φ = Φcr = 50%ACN

1.80 1.85 1.90 1.95 2.00 2.05 2.10 2.15 2.20 2.25 2.30 2.35 2.40 2.45 2.50 2.55 2.60 Minutes tliq tliq + Δt(q) Barrier Techniques 10

• Liquid Chromatography under Limiting Conditions (D. Berek, et al)

 of adsorption (LGA): Φsample > ΦCR, Φeluent < ΦCR

 of solubility (LCS): Φsample > ΦSOL, Φeluent < ΦSOL

 of desorption (LCD): Φsample < ΦCR, Φeluent > ΦCR

• Limitations: local solvent gradient depends on viscosity, column hydrodynamics, chromatographic conditions (flow rate, injection volume, concentration), separation is limited to column volume

• SEC-Gradients method (W. Radke)

Let’s discuss after Radke’s talk SEC vs. gradient IPC 11 Chromatographic characterizations of polymer blend: polystyrene / styrene-acrylonitrile copolymer / styrene-butadiene copolymer SEC: separation by size Gradient IPC: MW-independent elution

100 % Polystyrene

Φ 90

70 Eluent Gradient

60 SBR

Eluent Composition, St-Ac 50

14 15 16 17 18 19 20 21 22 23 24 25 26 27 0 2 4 6 8 10 12 14 16 18 20 Minutes Minutes Isocratic separation by size in THF at SEC column set Gradient separation by composition in ACN-THF gradient at C18 RP column Different modes of gradient separation 12 ® Elution of narrow polystyrenes at Symmetry C18 ACN – THF gradient (0 -100%) over 10 min Gradient Elution (GE) LAC oligomers 9,100 at CPA 50 Φ CR ΦCR = 48% THF 43,900 40 Eluent gradient 190,000 40 (at elution}

(THF,%) 355,000

Φ 710,000 Φ 30 1,260,000 30 2,890 20 20

10 10

Eluent Composition

0 0 3 4 5 6 7 8 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 Elution time, min (flow rate 1 ml/min) Log M Practical advice: even if you want to use LCCC, run gradient first: gradient elution is the best way to find critical point of adsorption

Brun, Alden, J. Liq. Chrom 2002 Solvent gradient methods 13

LAC GE at critical point of adsorption (CPA) separation by MW, chemistry, etc. separation by chemistry, topology Steric Steric

CPA CPA redissolution

Adsorption Precipitation Adsorption Precipitation

Precipitation-redissolution chromatography (elution at dissolution point) (GPEC) separation by MW and chemistry Steric

CPA does not exist

redissolution

Adsorption Precipitation

Different modes of gradient separation 14

Elution of narrow polystyrenes at Nova-Pak® C8

Gradient elution at Gradient elution at CPA redissolution point (GPEC)

100 100

80 80 ΦCR

60 Eluent gradient 60 (%THF) Φ Eluent gradient 40 40

20 20

0 0 14.0 14.5 15.0 15.5 16.0 16.5 17.0 17.5 18.0 18.5 19.0 19.5 14.0 14.5 15.0 15.5 16.0 16.5 17.0 17.5 18.0 18.5 19.0 19.5 Elution time, min ACN – THF (0 -100%) over 10 min MeOH – THF (0 -100%) over 10 min Φ does not exist: elution at redissolution Φ*sol < ΦCR: elution at CPA CR Brun, Alden, J. Liq. Chrom 2002 Different modes of gradient separation 15

Elution of polystyrenes at Nova-Pak® C8

X – THF (0 -100%) over 10 min 63 Elution at redissolution (%THF)

g 53 Φ ΦCR

43 Elution at CPA X: MeOH ( ) - elution at redissolution ACN ( ) –elution at CPAn 33 LAC

3 2.0 4.0 6.0 8.0 Log M

Only elution at CPA is MW-independent! Gradient elution at redissolution point (GPEC) 16

Liquid-liquid phase transition in the presence of solid surface: ∆F ∼ M (Φ – Φ*sol)

Usually, Φ*sol > Φsol

0.8 Φinj < Φ*sol * Φ sol Gradient elution at CPA can occur only if Φ*sol < ΦCR 0.6 Otherwise, ΦCR does not exist and elution is MW-dependent 0.4

3 5 7 Log M

Threshold depends on polymer molecular weight and concentration (Armstrong et.al., 1984) How to control the transition from LAC to GE at CPA 17

Transition point: Q ~ 1 Q > 1 (CPA) ΦCR

Q < 1 - retention depends on molar mass M (LAC)

Φ0

–1 0 1 2 Log Q 2Rg dε Q = X X Φ′ a D dΦ Transition to CPA occurs faster (at lower M) for: Rg - radius of gyration  narrow pores (low D) D - pore diameter  steep gradient (high Φ′) ε - segment interaction energy  high selectivity (high dε / dΦ) Φ′ - gradient rate Effect of operating conditions 18 ® Elution of narrow polystyrenes at NovaPak Silica Hexane – THF gradient (0 -100%) over 10 min 45 63

ΦCR 40 53 Theory vs. Experiment 35 Gradient time: 43 (%THF) (%THF)

g 30

Φ 5 min 33 10 min 25 15 min 23 20 20 min 25 min 13 15 30 min

10 3 3.5 4.0 4.5 5.0 5.5 6.0 Log M

Effect of gradient rate: shallower gradient – later transition to CPA Temperature gradient interaction chromatography (TGIC) 19 • IPC at adsorption conditions (LAC): separation by M, not size, but for oligomers only

• ΦCR depends on T, but dε /dΤ << dε/dΦ

• Temperature gradient should be much more effective in separation by MW

TGIC Steric

T > TCR

TCR CPA T0 Φisocr = Φ(TCR)

Adsorption Precipitation

Temperature vs. solvent gradients TGIC of narrow polystyrenes at Symmetry® C18

T’ = 2°C/min, flow rate 1 ml/min, Φisocr = 44% THF, TCR = 60C

20 18 Start temp.:

16 T0 = 40°C 14

10 T0 = 60°C

6 Solvent retnetion time [min] retnetion 4 gradient,

2 T=60C

0 3 4 4 5 5 6 6 log M

• Able to resolve high M species • Solvent gradient is still useful to identify critical conditions • Intrinsic relation between solvent & temperature gradient • TGIC could be coupled to SEC in 2D setting: SEC-IPC for branching analyses

October 8, 2014 DuPont Confidential 20 Gradient elution at CPA 21 Applications • Separation of polymer blends • Separation of telechelic polymers by functional groups • Separation of copolymers by chemical composition and microstructure, e.g. Blockiness • Copolymer purification by flesh chromatography based on IPC • Separation of nanoparticles by surface chemistry Benefits • Easiest way to find CPA • Broad range of eluents (including non-solvents) • Practically no limitation on MW • Superb resolution, especially at narrow pore columns • No peak splitting or some other chromatographic artefacts Potential problems • Break-through effect • Multiple interaction mechanisms for complex polymers • Competition with dissolution mechanism of retention for non-solvents

Selectivity of gradient elution at CPA 22

® Separation of poly(alkyl methacrylate) blend (MW ~ 300K) at Symmetry C18

ACN – THF gradient (0 – 100%) over 30 minutes

24 PnHMA PEMA PnBMA 20 PLMA

PMMA 8

4 0 5 10 15 20 25 Elution time, min

Brun, Alden, J. Liq. Chrom 2002 Efficiency and resolution on narrow pores (D ~ 6 nm)23

6 ® Separation of ultra-high M ( > 10 ) polyacrylates at Nova-Pak C18 ACN – THF (0 -100%) over 10 min

PEA PiBMA 100 PMMA PEHA PnBA PnBMA

80 D << Acrylate Rg~50 nm copolymers 60 (core-shell) blend

20 Eluent (THF,%) Eluent composition 0

14 15 16 17 18 19 20 21 22 23 24 Elution time, min Only “flower” conformations contributes to retention Brun, Alden, J. Liq. Chrom 2002 24 Separations by end-groups: isocratic vs. gradient OH Cl 4-arm PEGs with OH- and Cl-ends

Cl Water-ACN at XTerra® C18 OH Isocratic elution (LCCC) Gradient elution

water/ACN (60.1/39.9, v/v) water – ACN gradient: 20%ACN -100%ACN 700 0 350 0 600 number of chlorine ends 300 4 500 2 number of chlorine ends 250 400 mV 200mV 300 150 2 4 3 200 1 3 100 1 100 50

3 4 5 6 7 8 9 21 22 23 24 25 26 27 28 Minutes Minutes IPC-MS w/LTQ_Orbitrap: 4-arm PEG 25

Theoretical prediction of gradient elution of PEGs with various end-groups

Peak5 Peak4 4 Peak1 Peak2 Peak3 3 2 1

NL: TIC 1.55E8 TIC MS 052711_PE G_102_2_in _water_07 0 2 Cl 1 Cl 3 Cl 0 Cl 4 Cl

30 32 34 36 38 40 42 44 46 48 50 52 Time (min) Gradient elution for copolymer characterization 26

• Critical point of adsorption for statistical copolymers

• Separation by chemical composition (chemical composition distribution)

• Separation by microstructure (blockiness)

IPC of copolymers (theory) 27

• Statistical copolymer  Contain a single CPA (like in homopolymer)  CPA of statistical copolymers depends on chemical composition and microstructure (blockiness) of polymer chains • Block-copolymer  Does not contain a single CPA, but each individual block has its own CPA  Retention in gradient elution depends on MW and composition  Could be separated by block-length in gradient mode

• Effect of blockiness on retention in gradient elution  Always increases retention  Elution time at same chemical composition: alternating < statistical

• Practical implication  Gradient elution allows for separation by chemical composition and microstructure

Y. Brun, J. Liq. Chrom., 1999 Y. Brun, P. Foster, JSS, 2010 Isocratic elution of chlorinated polyethylenes (38% Cl)28

Eluent compositions Φ (CHCl3, %)

4.8 ΦCR= 40.5% 36

M 38

4.6

Log 4.4 44 Silasorb 600 Silica, different hexane-CHCl3 compositions 4.2

4.0

3.8

4.6 0 5 10 15 20 25 30 35 40 45 Minutes

Chromatographically, statistical copolymers behave similar to homopolymers: they contain CPA

Y. Brun, J. Liq. Chrom., 1999 RAFT polymerization of 2-EHA/n-BA (50/50) statistical copolymers29

CH2 CH C O O CH2 CH2 CH2 CH3

CH2 CH

CH2 H3C

CH2 CH

O O

CH3 5 3 4 6 7 2 1 3.0 1 8 Sample Mn Mw PD Conv ( H-NMR) Stat-1 6450 7278 1.13 <1 Stat-2 8694 9617 1.11 <1 2.0 Stat-3 11585 12638 1.09 3

dwt/d(logM) Stat-4 13240 14657 1.11 5

1.0 Stat-5 14540 15856 1.09 12 Stat-6 15289 16734 1.09 73 Stat-7 16065 17704 1.10 89 0.0 Stat-8 17107 18918 1.11 95 3.4 3.6 3.8 4.0 4.2 4.4 4.6 Log M Isocratic elution of statistical 2-EHA/n-BA copolymers 30

C18, different ACN-THF compositions Critical Point of Adsorption (CPA) Elution at different compos. Φ (THF,%):

THF-ACN (60/40) 100 70 60 58 56 54 1 - 8 4.3

ΦCR, stat = 60%THF 4.2

4.1

Log M 3.9

tliq 3.8

1.0 2.0 Vliq 3.0 4.0 0 4 5 6 7 8 9 10 11 12 13 14 15 Elution volume Ve (mL) Elution time, min (flow rate 0.25ml/min)

Gradient elution of statistical 2-EHA/n-BA copolymers 31

® XTerra C8, ACN – THF (0 -100%) over 10 min

70 100 8 90 ΦCR,stat 80 60 (THF,%)

70 5,6,7 Φ Φ 60 CR,stat 50 3 50 Eluent gradient

2 at Elution 40 40 PEHA (42K) Φ 30 4 1 20 PBA (33K) 30 10 Eluent Composition Composition Eluent 0 20 17.0 17.5 18.0 18.5 19.0 19.5 20.0 20.5 21.0 21.5 3.8 3.9 4.0 4.1 4.2 4.3 Elution time, min (flow rate 1 ml/min) Log M Φ = 42% CR, PBA Effect of MW on retention ΦCR,stat = 60% ΦCR,PEHA = 68% Gradient elution: theory vs. experiment 32

Stat. 2-EHA/n-BA copolymers at different C18 columns

ACN – THF (0 -100%) at different gradients

Effect of Gradient Rate Effect of Pore Size 60 67 65 55 63

50 61 % THF) at ElutionTHF) % % THF) at ElutionTHF) % ( ( 59 Φ 45 Φ 57

40 55 3.8 3.9 4.0 4.1 4.2 4.3 3.8 4.0 4.2 4.4 Log M Log M Column: XTerra ® 127A Columns: Symmetry ® 300A Gradient time: 5 min XTerra ® 127A 10 min Nova-Pak ® 60A 15 min Gradient time: 10 min 20 min Chemical composition heterogeneity by gradient elution 33

® Ethylene-Methyl Acrylate Copolymers at Nova-Pak C18 mobile phase gradient: EtAc/MeOH (50/50)-toluene (50 - 100%) over 10 min

340 Narrow chemical composition distribution 300 (autoclave)

260

mV 220

180

140 Broad chemical composition distribution 100 (tubular reactor) 60

12 13 14 15 16 17 18 19 20 21 Elution time (min)

Y. Brun, M. Pottiger, Macrom. Symp., v. 282, 2009 RAFT polymerization of A/B block-copolymers 34

CH2 CH C O O CH2 CH2 CH2 CH3 A (2-EHA)

CH2 CH

CH2 H3C

CH2 CH

O O B (n-BA)

CH3 2 4 1 3 5 6 7 8 Sample Mn Mw PD % 2-EHA 3.00 Block 1 (Homopol A) 7117 7786 1.094 100.00

Block 2 7548 8569 1.135 91

2.00 Block 3 8482 9660 1.139 81

Block 4 9256 10629 1.148 73 dwt/d(logM) 1.00 Block 5 10169 11811 1.161 66

Block 6 11293 13666 1.210 57

Block 7 11801 14585 1.236 53 0.00 Block 8 12127 15473 1.276 50 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8

Log M Gradient Elution of Poly(2-EHA-block-n-BA) 35

® XTerra C8, ACN – THF (0 -100%) over 10 min

5 4 100 3 2 1 PBA PEHA 6

80 7 ΦCR, PEHA (THF,%)

8 Φ 60 Eluent gradient

ΦCR, PBA 40

20 Eluent Composition Composition Eluent

0 17.0 17.6 18.2 18.8 19.4 20.0 20.6 21.0 Elution time, min

Composition affects retention stronger than MW Effect of copolymer microstructure on gradient elution 36

Statistical and block- 2-EHA/n-BA (50:50) copolymers

® , ® Nova-Pak Silica XTerra C18 Hexane – THF (0 -100%) over 10 min ACN-THF (0 -100%) over 10 min

100 100 PEHA PEHA PBA PBA

80 80 Stat. Block Block Stat. (Mn 12127) 60 (Mn 17107) 60

40 40 Eluent Composition (THF,%)

20 CPA depends on microstructure: separation20 by CPA = separation by blockiness Blockiness always increases retention

0 0 12.0 12.5 13.0 13.5 14.0 14.5 15.0 15.5 16.0 16.5 17.0 15 16 17 18 19 20 21 22 Elution time, min Effect of copolymer microstructure on gradient elution 37

Statistical and block styrene/MMA (50/50) copolymers

® ® Nova-Pak Silica, Nova-Pak C18 Hexane – THF (0 -100%) over 10 min ACN-THF (0 -100%) over 10 min

100 Block (93K) Block (407K) 100 Block (93K) Block (407K) PMMA Stat (91K)

PS (107K) PS 80 PMMA (103K) 80 Stat (91K)

60 60

40 40

Eluent Composition (THF,%) Retention of block-copolymers increases with MW 20 20

0 0 12 13 14 15 16 17 18 19 20 21 22 23 24 12 13 14 15 16 17 18 19 20 Elution time, min

Brun, Foster, JSS, 2010 Characterization of fluoro-containing aromatic polyesters 38 How to control microstructure of condensation copolymers?

F F CF3 CF3 3-GF16-iso F F O F F O F " onomer rs " F M fi t O H F F “Monomer first”: O O traditional copolycondensation + O O + HO OH O O should produce statistical copolymer n m O O

z Ty orcat - - F F MeOH, PDO CF3 “Oligomer first”: F CF3 F O F F O F chain extension in melt may produce blocky copolymers F O H F F if transesterification is suppressed by phase separation O O O O

O O O O n Chain-extended m 1.2 copolymer PTT F CF F CF F 3 3 1.0 3-GF16-iso F O F F " omer rs " O F Olig fi t F O H F 0.8 PTT F O O + O O 0.6 O O O O m n 0.4 z Ty orcat

- - F F weight fraction (1/log(g/mol)) differential MeOH, PDO CF3 F CF3 0.2 F O F F O F F O H F 4 5 F 1000.0 1.0x10 1.0x10 molar mass (g/mol) O O O O

O O O O N. Drysdale, Y. Brun, E. McCord, F. Nederberg, n m Macromolecules, 2012 Gradient elution of “monomer first” copolymers 39

FluoroFlash F8, water-HFIP Chemical composition calibration curve (MW independent) UV chromatograms gradient 65-100%HFIP AU 100 90 0.20 mol %F16–iso:

) 80 gradient 60-100%HFIP 100 % 0.16 10 (mol

60 Polynomial fit 0.1 2 40 50 0.08 25 copolymer in 20 iso

0.04 - 16 F

0.00 0

10 12 14 16 18 20 22 24 26 28 30 32 34 40 50 60 70 80 90 100

Minutes MP composition, HFIP%

Separation of statistical copolymers by chemical composition (elution at CPA) Chromatographic identification of copolymer microstructure 40

1.3 Blocky “oligomer first” 1.2 0.07 50 mol% 0.06 Statistical “monomer first” 1.1 50 mol% 1.0 0.05 PTT 0.04 0.9 AU 0.03 0.8 0.02 0.7 0.01 AU 0.6 0.00 0.5 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 0.4 Minutes 0.3 0.2 0.1 0.0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Minutes Purification based on step-wise flash IPC 41 Purification of St/MMA copolymer from residual homopolymers

1st elution 2nd elution

PS Copolymer PMMA removal removal removal window window window

Final solution (PMMA stayed on the column)

Intermediate solution (PS removed)

Initial solution (PS, PMMA, copolymer) 15 16 17 18 19 20 21 Minutes ELSD Chromatograms in Hexane-THF Gradient on Silica column Separating Nanoparticles by Surface Chemistry 42

Nanoparticle Free polymer chains grafted within the monolith pores provide higher degree of interaction between nanoparticle and column

Stationary Phase

• Monolith column produce better recovery for nanoparticles

• Rigid surface provides minimal tangential nanoparticle/column interaction

• Currently commercially available monoliths from styrene-divinyl crosslinked gel contain relatively short chains. Monoliths with grafted flexible chains are needed

• Recently done at the Molecular Foundry at Berkley National lab (Dr. Frantisek Svec)

Separation of decorated colloidal silica by degree of coating 43

Separation of colloidal silica coated with mix of phenyl- and aminophenylsilane

PhTMS / APhTMS ratio:

1:3 Mobile phase: toluene-DMAc gradient PhTMS Column: BMA/EDMA monolith grafted with PMMA chains (F. Svec, Berkley ) APhTMS 3:1 1:1

9:1 Si-OH

13 14 15 16 17 18 19 20 21 22 23 24 25 26 Elution time, min

• Long and flexible polymer chain functional groups (bonded phase) produce “reversed” critical point of adsorption. • Nanoparticle is “swallowed” by these chains through multiple attachment mechanism First reported at HPLC 2012 44