CONTEMPORARY LIQUID CHROMATOGRAPHY OF POLYMERS Event/201… · size-exclusion chromatography Average pore size 130 Å . 0 5 10 15 20 25 30 35 40 45 50 0 0.5 1 1.5 2 2.5 3 H (µ m)

CONTEMPORARY LIQUID CHROMATOGRAPHY

OF POLYMERS

PETER SCHOENMAKERS

IF POLYMERS WERE

ANIMALS

A typical polymer

A stressed polymer

Deborah Debbie

A confined polymer

Lamda

A congressional polymer

Polymers don’t move much (they just wobble a bit)

The relatively small polymer moves faster than the very big one

Polymers may take (seemingly) forever to move in and out of confined spaces

Polymers are claustrophobic

They are happy in a big hole



They don’t mind small holes



They don’t mind small holes

They don’t like to sit tight

Column band broadening in size-exclusion chromatography Average pore size 130 Å

0

5

10

15

20

25

30

35

40

45

50

0 0.5 1 1.5 2 2.5 3

H(µ

m)

u (mm/s)

toluene (ca. 4A)PS 5 kDa (33 A)PS 10 kDa (49 A)PS 20 kDa (73A)PS 30 kDa (93 A)PS 52 kDa (126 A)

Column band broadening in size-exclusion chromatography Average pore size 130 Å

Similar results were observed by F. Gritti and G. Guiochon, Anal. Chem . 79 (2007) 3188-3198

Higher Performance LC

The last decade has seen a paradigm shift in LC From high-pressure (or high-performance) HPLC To ultra-high-pressure or ultra(-high)-performance U(H)PLC

ul·tra [ˈʌltrə, uhl-truh] adjective going beyond what is usual or ordinary;

excessive; extreme.

ul·tra [ˈʌltrə, uhl-truh] adjective going beyond what is usual or ordinary

excessive; extreme. HYPERFORMANCE

LC

H - HETP (plate height) A - eddy-diffusion coefficient B - longitudinal diffusion coefficient u0 - linear velocity C - resistance-to-mass-transfer coefficient dp - particle size Dm - diffusion coefficient of analyte in mobile phase

uopt m

pmp D

udC

uBDAdH 0

2

0++=

The Van-Deemter Equation

Liquid chromatographers want to use smaller particles

Because these yield lower plate heights at higher velocities

HPLC U(H)PLC

Constant plate count (N) L ÷ dp

F ÷ dp-1

tR ÷ dp2

VR ÷ dp ∆P ÷ dp

-2

Shorter columns Much shorter analysis times Smaller eluent volumes Much higher pressures

Smaller particles are desirable

Higher pressures

needed

HPLC U(H)PLC


F ÷ dp-1

tR ÷ dp2

VR ÷ dp ∆P ÷ dp

-2

Much more heat generated Radial temperature gradients jeopardize performance Narrower columns are required for effective heat dissipation


Higher pressures

needed

HPLC U(H)PLC


F ÷ dp-1dc

2

tR ÷ dp2

VR ÷ dpdc2

∆P ÷ dp-2

Drastic reduction in extra-column volumes needed

About a factor 10 when comparing with 3 µm

particles or a factor 15 in comparison with 5 µm

particles


Higher pressures

needed

Narrow columns needed

HPLC U(H)PLC


F ÷ dp-1dc

2

tR ÷ dp2

VR ÷ dpdc2

∆P ÷ dp-2

Drastic reduction in extra-column volumes needed

Design of columns, connectors and instruments is especially critical for (slowly diffusing!) polymers


Higher pressures

needed

Narrow columns needed

Size-based separations

Both reducing the tubing length and increasing the column volume are needed to obtain acceptable extra-column dispersion

The observed peak should reflect the actual molecular-weight distribution of the polymer the contribution of the sample dispersity (PDI)

should exceed 90% for reliable characterization!

2222columnextracolumnPDIobserved −++= σσσσ

Extra-column contribution (%) for different system configurations

PS molecular weight, Da

2.1 x 50 mm column, using

Column Manager (CM)

2.1 x 50 mm column tubing

length reduced

(avoiding CM)

2.1 x 150 mm column,

tubing length reduced

(avoiding CM)

4.6 x 150 mm column,

tubing length reduced

(avoiding CM)

two 4.6 x 150 mm columns, tubing length

reduced (avoiding CM)

92 (toluene) 87 73 42 6 31990 95 51 16 2 0.5

30320 90 37 14 3 1.552400 94 55 22 6 3523000 102 68 35 12 62061000 90 39 12 12 7

Awkward compromise

Heat dissipation High pressures imply much heat. Narrow columns are required to avoid radial gradients (and excessive band broadening) Slow diffusion Wide-bore columns are required to avoid excessive extra-column band broadening For polymers the best compromise may be 4.6 mm i.d. Polymer detectors (light scattering, viscometry) for UHPLC or not yet available

HPSEC Columns: 3×250 mm (10-µm particles) 7.5 mm i.d. PL-Gel Mixed B Mobile phase: THF 1 mL/min

UPSEC Column: 150 mm (1.7-µm particles ) 4.6 mm i.d. Acquity UPLC BEH C18 Mobile phase: THF 0.5 mL/min

1270 1370 1470 1570 1670t (sec)

1.8 2 2.2 2.4 2.6 2.8 3t (min)

Size-exclusion chromatography of polyurethanes

10 times faster 20 times less eluent much better resolution

YES, WE CAN! Perform HP-SEC separations Perform UHP-SEC separations

The pace of change

in 30 min

in 3 min

1.5

2.5

3.5

4.5

5.5

6.5

7.5

0.12 0.14 0.16 0.18 0.2 0.22 0.24 0.26

log

M

Velution, mL

Hydrodynamic chromatography 1.7-µm particles

SEC pore sizes 70 – 300 Å

UPSEC Calibration curve

PS standards; Mobile phase THF, 0.5 mL/min Column Acquity UPLC C18, 100 × 2.1 mm I.D.

1.5

2.5

3.5

4.5

5.5

6.5

7.5

0.12 0.14 0.16 0.18 0.2 0.22 0.24 0.26

log

M

Velution, mL

SEC pore sizes 70 – 300 Å

UPSEC Calibration curve

PS standards; Mobile phase THF, 0.5 mL/min Column Acquity UPLC C18, 100 × 2.1 mm I.D.

Other columns are now also available for UPSEC

Petra Aarnoutse et al. – SCM-7

Molecular-weight limit is determined by pore size of stationary phase

Fast size-based UPLC polymer separations

Separation in the SEC region column 150 x 4.6 mm, flow rate 1.85 mL/min,

Pressure 660 bar (system limit at this flow rate)

AU

0.000

0.010

0.020

0.030

0.040

0.050

0.060

Minutes 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80

toluene PS 980

PS 2970 PS 7000 PS 19,880

PS 52,400

Molecular weight limit is determined by onset of molecule deformation

Separation in the HDC region column 150 x 4.6 mm, flow rate 1.85 mL/min,

Pressure 660 bar (system limit at this flow rate)

PS 52 kDa

AU

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

Minutes

0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 0.65 0.70

PS 96 kDa PS 197 kDa

PS 523 kDa PS 1,112 kDa

6 s 20 s

E. Uliyanchenko et al. J. Chromatogr. A 11 (2011) 1508

Polymers are subject to stress

y

x

Shear rate = γ = dvx/dy

Effect of particle size

vx,opt ÷ dp-1

y ÷ dp

γ ÷ dp-2

Stress

The extent of stress can be described by Deborah numbers (De). Polymers may undergo flow-induced stretching during HDC separations The transition from coil to stretched state of a polymer occurs around De = 0.5

Polymer deformation

D. Hoagland, R. Prud’homme. Macromolecules 1989, 22, 775-781 skip

Where: k – constant which depends on the packing structure; v – superficial solvent velocity (flow per unit area of empty bed) dp – particle diameter Φ – Flory-Fox parameter (2.5·1023 mol-1) η – solvent viscosity rG – radius of gyration of the polymer R – gas constant T – temperature

Deborah number

RTr

dv G

pkDe

312.6 ηΦ⋅⋅=

Shear Rate

Size of the molecule

Samples: PS in THF, Column ID 2.1 mm, dp 1.7 µm, Temperature 250C

Deborah numbers (deformation above 0.5)

Polymer MW, DaFlow rate, mL/min

0.1 0.2 0.5 0.7 11,000,000 0.05 0.11 0.27 0.38 0.542,000,000 0.18 0.37 0.92 1.3 1.83,000,000 0.38 0.75 1.9 2.6 3.89,000,000 2.6 5.2 13 18 26

Very-high-molecular-weight polymers may be deformed under UHPLC conditions

Slalom chromatography (SC) – a tunnel effect

Smallest polymers elute first Large polymers elute (much) later Large polymers must adapt their shapes

Hydrodynamic chromatography Coiled molecules

Size-exclusion chromatography

Slalom Chromatography Stretched molecules

Mcrit Coil-stretch transition

PS standards. Column Acquity UPLC C18, 1.7-µm particles, pore sizes 70 – 300 Å, 100 × 2.1 mm I.D., Mobile phase THF, 0.5 mL/min

UP size-based separations – Calibration curve

1.5

2.5

3.5

4.5

5.5

6.5

7.5

0.12 0.14 0.16 0.18 0.2 0.22 0.24 0.26

log

M

Velution, mL

Two peaks for one “narrow” PS standard

1.5

2.5

3.5

4.5

5.5

6.5

7.5

0.12 0.14 0.16 0.18 0.2 0.22 0.24 0.26

log

M

Velution, mL

The first peak emphasizes the low-molecular-weight fraction Application: Polymer degradation studies

Stress resistance of polymers?

Deformation (reversible)

Degradation (irreversible)

PS standard (Mr = 3 MDa) injected on UPLC column 100 mm × 2.1 mm i.d.; 1.7-µm particles at different flow rates 0.1 mL/min (De=0.4) 0.2 mL/min (De=0.8) 1 mL/min (De=3.9)

Peaks were collected and re-injected at 0.1 mL/min

The obtained peaks were compared to the PS standard of similar concentration, which was not previously injected on the column

Degradation study of polystyrene 3 MDa

Degradation study of polystyrene 3 MDa 1. Eluted from UPLC 100 mm × 2.1 mm i.d. column (1.7 µm particles)

at different flow rates - 0.1 mL/min (De = 0.4)

- 0.2 mL/min (De = 0.8) - 1.0 mL/min (De = 3.9) 2. The peaks were collected and re-injected at a flow rate of

0.1 mL/min 3. The obtained peaks were compared to the original PS standard

PS

Degradation products of THF?

5 mL/min on 4.6-mm i.d. column 15 mL/min on 8-mm i.d. column

- 0.1 mL/min (De = 1.9) - 0.3 mL/min (De = 5.6) - 0.5 mL/min (De = 9.3) - 0.7 mL/min (De = 13.1) - 0.9 mL/min (De = 16.8)

Polymers re-injected at 0.1 mL/min

0.1 ml/min

0.3 ml/min 0.5 ml/min

0.7 ml/min

0.9 ml/min

Slalom Chromatography Y. Liu, W. Radke, H. Pasch. Macromolecules 2005, 38, 7476-7484


1.7-µm particles 2.1-mm i.d. column

- 0.1 mL/min (De = 5.1)

- 0.5 mL/min (De = 25.6)

PS standards re-injected at 0.1 mL/min. Normalized chromatogram

0.5 ml/min

Not injected before

0.1 ml/min


Evidence of chain degradation of very large PS standards (> 3 MDa) (using very small particles and very high velocities, De > about 5)

1.7-µm particles 2.1-mm i.d. column

Pressure-driven separation

Channel of molecular dimensions

Increased retention for branched polymers requires low flow rates and very narrow channels (Size of molecules, Rh > 0.4 Rchannel)

Rh

Molecular-topology fractionation (MTF) Branching-selective separation in very narrow channels*

Polymeric monoliths

* Meunier D.M., Smith P.B., Baker S.A., Macromolecules 38 (2005) 5313

skip

MTF×SEC of linear polystyrene standards

at 10 µL/min

1.3 2.6

3.7 MDa

Critical MTF×SEC of linear polystyrene standards at 30 µL/min

1.3 2.6 3.7 MDa

Critical MTF×SEC of “star-shaped” polystyrene

Linear PS

(2.6 MDa)

One-arm star (3.9 MDa)

Two-arm “star”

(5.2 MDa)

One single branching point in almost 40,000 monomeric units!

At 30 µL/min this implies three hours

Classification of separation methods λ = (radius of polymer)/(radius of flow-through channel)

Polymers are religious

They hide from the devil ...

... and fly with the angels

weak eluent / poor solvent

strong eluent / good solvent

S-co-MMA (60)

S-co-MMA (40)

S-co-MMA (80)

S-co-MMA (20)

PS 200,000

PS 2,450

PS 7,000

PS 30,000

PS 900,000

PMMA 127,000

PMMA 840,000

PMMA 28,300

PMMA 6,950

PMMA 2,990

Chemical composition

Mol

ecul

ar s

ize

LCxLC (LCxSEC) of religious polymers

UHPLC×UHPLC setup

From Column 1 To Column 2

Waste

Loop 1

Loop 2 Pump 1 Pump 2

Column 1 Column 2

Detector Detector

2D pump

PMMA 65 k

PMMA 50 k

PMMA 24 k PMMA 14 k

PMMA 100 k

PMMA/PBMA 80/20 80 k





PBMA 18 k PBMA 57 k

PBMA 100 k

Chemical composition

Mol

ecul

ar s

ize

Sample - mixture: -poly(methyl-methacrylate) -poly(n-butyl-methacrylate) -poly(methylmethacrylate)-block-poly(n-butyl-methacrylate)

Time, min 22 18 14 10 6

UPLC×UPSEC of polymers

Awkward compromise Heat dissipation High pressures imply much heat. Narrow columns are required to avoid radial gradients (and excessive band broadening)

Slow diffusion of polymers Wide-bore columns are required to avoid excessive extra-column band broadening

Pre-column band broadening Post-column band broadening

UPLC “General Solution” Gradient elution is routinely used for separating complex samples

Weak-eluting sample solvents (weaker than the starting eluent) allow focussing of the analytes at the top of the column

Injection band broadening can be negated and only detection band broadening pertains

Breakthrough effect in gradient-elution LC of polymers (PMMA 34,500)

0

10

20

30

40

50

60

70

4 5 6 7 8 9 10Time (min.)

ELSD

resp

onse

30µl

20µl

15µl

10µl

Column: C8-silica (250 x 4.6 mm i.d.; 5 µm particles; 100 Å pore size); Gradient: from 38% to 100% THF in water(25)/MeOH (75) in 20 min.

Skip explanation

Adsorption

Critical %

Below critical (adsorption conditions)

Above critical (exclusion conditions)

Exclusion Injection Plug

Adsorption peak

Explanation of breakthrough effect

time

“Breakthrough” peak

Adsorption

Direction of flow

Isocratic LC 73/30 n-hexane/THF Silica column

Breakthrough effect in isocratic LC of polystyrenes

breakthrough

adsorption

Isocratic LC 73/30 n-hexane/THF Silica column

Breakthrough effect in isocratic LC of polystyrenes

Avoiding breakthrough effects in gradient LC of polymers

Eva Reingruber et al., J.Chromatogr.A 1217 (2010) 6595-6598





Copolymers from ethyl and benzyl diazoacetate

O

O

OR1

OR1

OOR1

OOR1

nN2

O

R1ORhI-precatalyst

- N2t =

30 min

N2

O

R2O

- N2

O O

O O

OR1 OR2

OR1 OR1

OOR2

OOR2

n

homo block gradient block


c

a b

d

Copolymers from ethyl and benzyl diazoacetate

The more peaks the merrier?


Breakthrough in second-dimension SEC?

One-dimensional SEC (solvent CHCL3)

Projection from LC×SEC

t0

Effect of sample solvent in SEC

Sample solvent CHCL3 / MeOH 60/40

Sample solvent CHCL3

40 µL 50 µL

62.5 µL 87.5 µL

Effect of transfer volume in LC×SEC

40 µL 50 µL

100 µL 150 µL

Effect of transfer volume in LC×SEC

SEC×LC vs. LC×SEC

HR-SEC possible in 1D

Possible 2D focusing

Possible sample clean-up

2tR not limited

2D breakthrough

2D gradients

1D overloading, adsorption

Limited choice of detectors

HR-gradient LC possible

Choice of detectors

2tR limited

Independent optimization

High sample capacity

Low-resolution 2D SEC

Possible 1D breakthrough

Possible 2D adsorption

Align separation selectivity with sample dimensions Excellent separations Structured, easily interpretable chromatograms Need to avoid 2D injection band broadening / breakthrough 1D effluent should not be too good a 2D eluent Need to avoid 2D adsorption 1D effluent should not be too bad a 2D eluent When using gradients the 1D effluent varies in time

LC×LC of polymers

SEC×LC vs. LC×SEC

HR-SEC possible in 1D

Possible 2D focusing

Possible sample clean-up

2tR not limited

2D breakthrough

2D gradients

1D overloading, adsorption

Limited choice of detectors

HR-gradient LC possible

Choice of detectors

2tR limited

Independent optimization

High sample capacity

Low-resolution 2D SEC

Possible 1D breakthrough

Possible 2D adsorption

Align separation selectivity with sample dimensions Excellent separations Structured, easily interpretable chromatograms Need to avoid 2D injection band broadening / breakthrough 1D effluent should not be too good a 2D eluent Need to avoid 2D adsorption 1D effluent should not be too bad a 2D eluent When using gradients the 1D effluent varies in time

LC×LC of polymers – major dilemma

We need to switch solvents

Fist-dimension peak profile

“Passive modulation” (as in LC×LC)

“Active modulation” (as in GC×GC)

Modulation

Loop

1

Detector

Waste

Injector

Pum

p 1

Pump 2 Lo

op 2

Col

umn

1

Col

umn

2

Column-based LC×LC

Loop

1

Detector

Waste

Injector

Pum

p 1

Pump 2 Lo

op 2

Col

umn

1

Col

umn

2

SPE

1 Detector

Waste

Injector

Pum

p 1

Pump 2

SPE

2

Col

umn

1

Col

umn

2

Column-based LC×LC with SPAM Stationary-phase assisted modulation

SPE

1 Detector

Waste

Injector

Pum

p 1

Pump 2

SPE

2

Col

umn

1

Col

umn

2

HYPERformance LC

HYPERFORMANCE

LCAndrea Gargano Michelle Camenzuli Anna Baglai Henrik van de Ven (Rudy Vonk) (Margaryta Ianovska) SCM-7

Conclusions

Despite some complications, UHPLC can have significant benefits for the separation of polymers SEC (Mr < 50 kDa PS) and HDC (Mr < 1 MDa PS) separations may be performed Very large molecules (Mr > 1 MDa PS) may be deformed, resulting in slalom chromatography Ultra-high-molecular-weight polymers (Mr > 3 Mda PS) may degrade in the column at high flow rates LC×LC is indispensable for polymer characterization Perhaps LC×LC can be improved using active modulation

Martin Lopatka (NWO) John Mommers (DSM)

THE END

Analytical-Chemistry / Forensic-Science Group I S

CM

SCM-7 Seventh International

Symposium on the Separation and Characterization of

Natural and Synthetic Macromolecules Amsterdam, The Netherlands

January 28-30, 2015

www.scm-7.nl

Documents

CONTEMPORARY LIQUID CHROMATOGRAPHY OF POLYMERS Event/201… · size-exclusion chromatography Average pore size 130 Å . 0 5 10 15 20 25 30 35 40 45 50 0 0.5 1 1.5 2 2.5 3 H (µ m)