André M. Striegel · 2017. 2. 17. · quadruple-detector SEC. ... Base pair sequence Genetic code,...

Copolymer Characterization

by

Multi-Detector

Size-Exclusion Chromatography

André M. StriegelNational Institute of Standards & Technology

Objectives

• Introduction to macromolecular distributions.

• Determine chemical heterogeneity in copolymer using

quadruple-detector SEC.

• Examine and correct effect of n/c heterogeneity on

calculated M averages, distribution, etc.

• Extend method to “dual-absorbing” copolymers.

• Apply quintuple-detector SEC to show

physicochemical basis for conformational changes

across MMD.

Styrene Polystyrene

n can take on a range

of values

Therefore, so can M

10 20 30 40 50 60 70 80 900

20

40

60

80

100

120

Dif

fere

nti

al

We

igh

t F

rac

tio

n

Molar Mass

Molar Mass

(MM)

Short-Chain

Branching

(SCB)

Long-Chain

Branching

(LCB)

Polyelectrolyte

Charge

(+/-)

+

+

++

+

++

+

++

+

Chemical

Heterogeneity

(CH)

Chemical

Composition

Distribution

(CCD)

MM

SCB

LCB

+/-

CH

Ch

em

ica

l Co

mp

os

ition

Differential Weight Fraction

CCD

Striegel, Yau, Kirkland, & Bly, Modern Size-Exclusion Liquid Chromatography, 2nd ed. (2009)

Macromolecular distributions: Measurement and End-Use Effects

Macromolecular Representative end-use Separation method used for

Property properties affected determination

Molar mass Elongation, tensile strength, adhesion SEC, FFF, HDC, TGIC, CEC, SFC, MALDI-MS,

rheology

Long-chain branching Shear strength, tack, peel, crystallinity SEC-MALS, SEC-VISC, rheology, enzymology

Short-chain branching Haze, stress-crack resistance, crystallinity SEC-IR, SEC-NMR, TREF, CRYSTAF, enzymes

Crosslinking Gelation, vulcanization, surface roughness SEC-MALS, SEC-VISC, rheology

“Architecture” Flow modification, diffusion, encapsulation SEC-MALS-QELS-VISC

Tacticity Crystallinity, anisotropy, solubility SEC-NMR, TGIC, LCCC,

Chemical composition Morphology, miscibility, solubility GPEC, TGIC

Chemical heterogeneity Toughness, brittleness, biodegradability SEC-spectroscopy/spectrometry, LCCC, PFC

Chemical composition Mechanical properties, blending, plasticization 2D-LC (e.g., SEC-GPEC)

vs. molar mass

Block sequence Dielectric properties, reactivity, miscibility SEC-spectroscopy, 2D-LC (e.g., PFC-SEC)

Base pair sequence Genetic code, heredity, mutations Automated DNA sequencing, MALDI-MS

Polyelectrolyte charge Flocculation, transport, binding of metals SEC-conductivity

Particle size Packing, drag, friction, mixing FFF, HDC, PSDA, sieving

Striegel, Yau, Kirkland, & Bly, Modern Size-Exclusion Liquid Chromatography, 2nd ed. (2009)

Measuring

Chemical Heterogeneity

in a

Random Copolymer

Macromolecular Distributions:Measurement & End-Use Effects

• Property: Chemical Heterogeneity

0

20

40

60

80

100

120

Dif

fere

nti

al W

eig

ht

Fra

cti

on

Molar Mass

Molar Mass

(MM) MM

CHChemical

Heterogeneity

(CH)

Relative ab

un

dan

ce

Macromolecular Distributions:Measurement & End-Use Effects

• Property: Chemical Heterogeneity

• Definition: Change in average chemical

composition of a copolymer as f(M),

uncorrected for local polydispersity.

Macromolecular Distributions:Measurement & End-Use Effects

• Property: Chemical Heterogeneity

• Affects: Melting point, gas permeation,

conductivity, interfacial fracture energy

• Measured using: SEC with physico-

chemical detection methods

Measuring Chemical Heterogeneity

• Random copolymer of styrene (St) and methyl methacrylate (MMA):

Is relative ratio of St:MMA constant as a function of molar mass?

• Styrene absorbs at 260 nm, but methyl methacrylate does not.

• UV detector @ 260 nm will respond only to styrene content in polymer.

• Differential refractometer will respond to both styrene and methyl methacrylate

in polymer.

Measuring Chemical Heterogeneity

DRIDifferential refractometer

UVUltraviolet

MALSMulti-angle light scattering

VISCDifferential Viscometer

Styrene (but not MMA)

absorbs @ 260 nm

Both St & MMA

elicit DRI response

Relative Detector Response:

DRI vs. UV

20 22 24 26

0.0

0.2

0.4

0.6

0.8

1.0

UV Signal Conc. of St only

Re

lati

ve

De

tec

tor

Sig

na

l (a

rbit

rary

un

its

)

Retention volume (mL)

DRI Signal Conc. of both St and MMA

iDRI

iUV

i

PSDRI

PSUV

PMMAPS

i

PS

PMMA

i

i

S

SZ and constant

S

SF where

c

n

c

nZ

c

nF

c

nZ

St

,

,

,

,

%100)(%

Haidar Ahmad & Striegel, Anal. Bioanal. Chem., 396 (2010) 1589

5.0x104

1.0x105

1.5x105

2.0x105

2.5x105

3.0x105

3.5x105

0.5

1.0

1.5

2.0

2.5

3.0

18

20

22

24

26

28

30

Dif

fere

nti

al W

eig

ht

Fra

cti

on

(d

Wf /d

M)

Molar Mass, M (g/mol)

MMD

% Styrene

Weig

ht P

erc

en

t Sty

ren

e (%

St)

Measuring Chemical Heterogeneity

Haidar Ahmad & Striegel, Anal. Bioanal. Chem., 396 (2010) 1589

Chemical Heterogeneity

Translates Into

n/c Heterogeneity

c

nn

c

n

c

0

0lim

n: refractive index of solution

n0: refractive index of solvent

c: concentration of analyte in solution

n/c versus %PS

y = 0.0011x + 0.0852

R2 = 0.9951

0.08

0.12

0.16

0.2

0 25 50 75 100% PS

dn

/dc

(mL

/g)

dn/dc vs % PS

Linear (dn/dc vs % PS)

75% PS Block

copolymer

100% PS

100%

PMMA

Alternating

copolymers

(50% PS)

20% PS random

copolymer

25% PS Block &

Random copolymers

What Does This Mean?

• Molar mass M determined by multi-detector SEC is

influenced by chemical identity of analyte.

• This influence enters through the n/c-dependence

of the static light scattering (SLS) and differential

refractive index (DRI) detector signals:

• M averages and distribution determined from ratio

of these signals at each SEC elution slice i:

cc

nM SSLS

2

c

ncSDRI

i

i

i

i

i

i

i

i

i

c

nM

cc

n

cc

nM

S

S

2

DRI,

,SLS

What Does This Mean?

• Therefore, chemical heterogeneity translates into

n/c heterogeneity.

• In turn, heterogeneity in n/c translates into a bias

in the molar mass averages and distribution

calculated via SEC/MALS/DRI .

• (This bias will also manifest itself in SEC using

LALS, peak-position calibrant-relative calibration, or

universal calibration).

We can correct for this:

• First, correct n/c for chemical heterogeneity:

iMMA

PMMA

iSt

PSicorrected

wc

nw

c

n

c

n,,

,

y = 0.0011x + 0.0852

R2 = 0.9951

y = 0.001x + 0.087

0.08

0.12

0.16

0.2

0 25 50 75 100% PS

dn

/dc

(mL

/g)

dn/dc vs % PS

Linear fit

Predicted

n/c = [(0.191 wSt) + [0.087 (1 - wSt)]

n/c versus %PS

We can correct for this:

• First, correct n/c for chemical heterogeneity:

• Then, use this value to correct calculated M:

iMMA

PMMA

iSt

PSicorrected

wc

nw

c

n

c

n,,

,

icorrected

duncorrecteiduncorrecteicorrected

c

n

c

n

MM

,

,,

Chemical Heterogeneity Corrected MMD

5.0x104

1.0x105

1.5x105

2.0x105

2.5x105

3.0x105

3.5x105

0.5

1.0

1.5

2.0

2.5

3.0

18

20

22

24

26

28

30

Dif

fere

nti

al W

eig

ht

Fra

cti

on

(d

Wf /d

M)

Molar Mass, M (g/mol)

Uncorrected MMD

Corrected MMD

% Styrene

We

igh

t Pe

rce

nt S

tyre

ne

(%S

t)

Uncorrected Corrected

Mn (g/mol) 170,000 11,900 175,000 11,900

Mw (g/mol) 200,000 800 209,000 800

Mz (g/mol) 234,000 2,100 250,000 2,100

Mw/Mn 1.18 0.08 1.19 0.07

Error in M depends both

on M and on

n/c difference among

monomers in copolymer

Haidar Ahmad & Striegel, Anal. Bioanal. Chem., 396 (2010) 1589

22

As % composition

changes…

[(n/c)PS × wSt] +

[(n/c)PMMA × wMMA](n/c)copolymer =

(n/ c)copolymer

changes

SDRI = c (n/c)copolymer

Concentration

changes

ccnMS 2SLS )/(

Molar mass

changes

Intrinsic viscosity

changes c

sp

c

lim

0

][

Solution

conformation

changes

Chemical Heterogeneity:

Effect on Calculated Parameters

Chemical Heterogeneity Also Affects

Calculated Conformation

7x104

105

2x105

3x105

4x105

50

100

150

Intr

insic

Vis

co

sit

y,

[]

(mL

/g)

Molar Mass, M (g/mol)

Uncorrected

Corrected

0.71 0.01

0.60 0.01

105

2x105

3x105

10

15

20

25

Ra

diu

s o

f G

yra

tio

n,

RG (

nm

)

Molar Mass, M (g/mol)

Uncorrected

Corrected

0.64 0.01

0.59 0.01

Mark-Houwink Plot Conformation Plot

Polymer adopts a less extended conformation in solution than originally believed.

Haidar Ahmad & Striegel, Anal. Bioanal. Chem., 396 (2010) 1589

Determining

Chemical Heterogeneity

in Copolymers:

The “Dual-Absorption” Case

“Dual-Absorption” Copolymers

• In previous case, only one monomer in copolymer absorbed at a given UV wavelength.

• What is both monomers absorb at the same wavelength, however?

• Also, what can adding QELS detection tell us about the copolymer?

• How does chemical heterogeneity affect solution structure?

• What is the underlying physicochemical basis?

Poly(AM-co-DMAM)

H2C CH CH2 CH

C O

NH2

C O

N

( () )x 1-x

Poly(acrylamide-co-N,N-dimethyl acrylamide)

Homopolymer Absorptivities

Absorptivity = 84 ± 1 mL ∙ mg-1 ∙ cm-1 Absorptivity = 1913 ± 1 mL ∙ mg-1 ∙ cm-1

@ 240 nm

Polyacrylamide

(PAM)

Poly(N,N-dimethyl acrylamide)

(PDMAM)

CH2 CH

C O

N

( )n

H2C CH

C O

NH2

( )n

SEC/MALS/DLS/VISC/UV/DRI

© Wyatt Technology Corporation 2005 - All Rights Reserved

What’s Next…

• Remainder of day

– hands-on experience with hardware and software

– batch lab experiment, real data!

• Summary session bring questions!

Solvent

Reservoir

Pump Injector Thermostatted

SEC columns MALS/DLS

DRI VISC

Fluid connection

Electronic connection

UV

Wt% DMAM at each SEC slice i

%

c

n

c

nSaaZ

aZc

nS

DMAM

PDMAMPAM

r,iR,CopolymePAMPDMAMPDMAM

PAMPDMAM

PAM

r,iR,Copolyme

i 100%

iDRI

iUV

iCopolymerRS

SS

,

,

,,

PDMAM

PDMAMPDMAMR

PDMAM

PDMAM

PDMAMDRI

PDMAMUV

PDMAMa

c

n

Sa

c

n

S

SZ

,

,

,

where

a = absoptivity of constituent

homopolymers

Rowland & Striegel, Anal. Chem., 84 (2012) 4812

Chemical Heterogeneity of

P(AM-co-DMAM)

Rowland & Striegel, Anal. Chem., 84 (2012) 4812

104

105

106

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Molar Mass, M (g mol-1)

Dif

fere

nti

al

We

igh

t F

rac

tio

n

72

74

76

78

80

82

84

86

88

90

92%

N,N

-dim

eth

yla

cry

lam

ide

Polymer Size(s)

• Radius of gyration (a.k.a. rms radius): From MALS + DRI

• Hydrodynamic (a.k.a. Stokes) radius: DLS (a.k.a. QELS) + DRI

• Viscometric radius: MALS + VISC + DRI

2/1

2)(1

1

i

c miG Rrn

R

Ts

BH

D

TkR

6

3/1

10

3

AN

MR

RG and RH Conformation Plots

Rigid Rod

Random Coil

Hard Sphere

0.60 ≥ α ≥ 0.50

α = 1

α = 0.33

105

106

1

10

100

Rad

ius (

nm

)

Molar Mass (g mol-1)

RH

RG

slope = 0.58

= 0.56

df = 1.8

Rowland & Striegel, Anal. Chem., 84 (2012) 4812

ρ ( ≡ RG /RH) Parameter

105

106

1

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8

1.9

2

R

G/R

H

Molar Mass, M (g/mol)

Linear Random CoilTransition to More

Extended Coil

PDMAM

homopolymer

Rowland & Striegel, Anal. Chem., 84 (2012) 4812

Mark-Houwink & Conformation Plots

Rigid Rod

Random

Coil

Hard

Sphere

0.80 ≥ a ≥ 0.50

a = 2

a = 0

105

106

1

10

100

10

100

1000

Rad

ius (

nm

)

Molar Mass (g mol-1)

a = 1.07

df = 1.45

a = 0.64

df = 1.8

[]

RH

RG

slope = 0.58

= 0.56

df = 1.8 In

trinsic

Vis

cosity

, [n] (m

L g

-1)

Rowland & Striegel, Anal. Chem., 84 (2012) 4812

Rη/RG Plot

105

106

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

R/R

G

Molar Mass, M (g/mol)

Linear Random CoilTransition to More

Extended Coil

PDMAM

homopolymer

Rowland & Striegel, Anal. Chem., 84 (2012) 4812

6x104

2x105

4x105

6x105

8x105

106

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

R

Molar mass, M (g/mol)

Poly(St-ran-MMA)

Poly(St-ran-MMA)

Poly(St-alt-MMA)

Poly(St-b-MMA), 75% St

Poly(St-b-MMA), 25% St

PS

PMMA

Hard-sphere limit

Appro

xim

ate

random

coil

ra

nge

RG

R/RG of Homo- and Copolymers

Haidar Ahmad, Striegel, & Striegel, Polymer, 52 (2011) 1268

Lack of Chemical Heterogeneity in

Random S-MMA Copolymers

Random Copolymer 126k

Random Copolymer 186k

Haidar Ahmad, Striegel, & Striegel, Polymer, 52 (2011) 1268

PS PMMA

S-SS-MMA MMA-MMA

1074 cm-1

IR peak

S and MMA Homo- and Copolymers

R/R

G

R/RG

R/RG

Fraction A Fraction B Fraction C

Area under

1074 cm-1

Area under

1074 cm-1

Area under

1074 cm-1< <

Molar mass (g/mol)

Off-Line IR Analysis of SEC “Heart-Cuts”

Persistence Length, Lp

Lpr

Î1

2/1

2

GR

M

1/M

2/1)3(2

3LpL MLM

2/1

3

p

L

L

M

222

31

32/12/1

2

Lp

Lp

p

L

GML

M for ,

M

ML

L

M

R

M

Striegel, Yau, Kirkland, & Bly, Modern Size-Exclusion Liquid Chromatography, 2nd ed. (2009)

Persistence Length of Poly(AM-co-DMAM)

3.0x10-6

4.0x10-6

5.0x10-6

6.0x10-6

7.0x10-6

32

34

36

38

40

42

(M/2LPM

L) = 2.2

Lp = 12 1 nm

ML = 3120 6 g mol

-1 nm

-1

(M/R

G2)1

/2

1/M

R2 = 0.993

Poly(AM-co-DMAM)

Rowland & Striegel, Anal. Chem., 84 (2012) 4812

104

105

106

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Molar Mass, M (g mol-1)

Dif

fere

nti

al W

eig

ht

Fra

cti

on

72

74

76

78

80

82

84

86

88

90

92

% N

,N-d

imeth

yla

cry

lam

ide

Transition in Polymer Conformation

Extended Coil

(“Alt”-like)

T

r

a

n

s

i

t

i

o

n

R

e

g

i

o

n

Random Coil

(“Homo”-like)

RG ≤ Lp Lp < RG ≤ 2Lp 2Lp < RG

Lp = 12 1 nmRowland & Striegel, Anal. Chem., 84 (2012) 4812

Info From Multi-Detector SEC(Not a Comprehensive List)

MALS

QELS

VISC

DRI

UV

[]

CC

M-H plot,

R, R/RG

M aves, MMD,

Confor. plot, Lp

CH

CC: Chemical compostion, CH: Chemical heterogeneity, M-H: Mark-Houwink, RG,z/RH,z

CONCLUSIONS

• Chemical heterogeneity in copolymers results in n/c heterogeneity.

• n/c heterogeneity can affect calculated M averages, distributions, etc.

• Appropriate choice of SEC detectors allows accurate characterization of copolymers.

• Can also inform knowledge of physicochemical basis for conformational changes as f(M).

• Method has also been extended to binary blends.