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Introductionto

SEC (GPC) Detection

André M. StriegelNational Institute of Standards & Technology

Why Do We Need Detectors in SEC?

We want to know what is eluting from our column.

We want to know when this takes place.

We want to determine certain physicochemical properties of our analyte(s).

We want to monitor system performance.

So…

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Course Module Objective

What detection method(s) do I need,and why?

What Detector(s) Do I Need, And Why?

To answer these questions, we need to know:

1. What property/properties of our analyte(s) do we want to determine (e.g., molar mass averages/distribution, long-chain branching, chemical composition, etc.)?

2. What level of quantitation, if any, do we desire?

3. Does a single detection method exist that can measure this property directly and quantitatively? If so, which method and how?

4. If not, can that information be obtained by using multiple detectors? If so, which methods and how?

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Should Be Aware Of…

Limitations of the separation method w/r to isolating property of interest. (Beyond objective of this module).

Limitations of the detection method(s) w/r to measuring and quantitating property of interest.

Today’s Menu

What detection methods are out there?

What do they measure?

How do some of them combine with each other?

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Classifying SEC Detectors

For purposes of discussion, we will classify SEC detectors as Chemical and Physical.

Chemical detectors provide information in an additive fashion.

Physical detectors provide information in a synergistic fashion

Some detectors can be either Chemical or Physical, depending on mode of operation or on how data are employed.

Chemical Detectors

UV

MS• ESI• MALD/I• ICP• IMS

IR/(Raman)

NMR

Fluorescence

Conductivity

Etc.

For the most part, these are methods you learned about in Instrumental Analysis (or related course) and function the way you were taught.

Fairly straightforward, when operated in Chemical mode (some can be Physical methods, as well).

Therefore, we will not spend much time on these.

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Chemical Detectors

For UV, IR/Raman, and fluorescence, need “active” (absorbent, fluorescent) groups on molecules.

For conductivity, need charge-bearing groups.

If these groups don’t exist, but you wish to use one or more of these detection methods, will need to derivatize(preferably quantitatively and w/o degradation) your polymer.

Chemical Detectors combine with each other additively.

SEC/UV SEC/IR SEC/FL

Kirkland & Antle, J. Chromatogr. Sci. 15 (1977) 137 Nomura et al., Macromolecules 35 (2002) 5388 Moon, Hoye, Macosko, J. Polym. Sci. A 38 (2000) 989

ex = 358 nm, em = 405 nm

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SEC/DRI/UV of St-MMA Copolymer

20 22 24 26

0.0

0.2

0.4

0.6

0.8

1.0

UV Signal Conc. of St only

Rel

ativ

e D

etec

tor

Sig

nal

(ar

bit

rary

un

its)

Retention volume (mL)

DRI Signal Conc. of both St and MMA

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

ctio

n (

dW

f /d

M)

Molar Mass, M (g/mol)

MMD % Styrene

Weig

ht P

ercent S

tyrene (%

St)

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

• 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.

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

SEC/ICP-MS

Precautions

Minimize use of organic solvents.

Careful attention to ionic strength of mobile phase.

Minimize salt content of mobile phase.

Careful sample preparation.

Zoorob, McKiernan, Caruso, Mikrochim. Acta 128 (1998) 145

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SEC/ICP-MS

Zoorob, McKiernan, Caruso, Mikrochim. Acta 128 (1998) 145 Wrobel et al., Anal. Chem. 75 (2003) 761

Physical Detectors Concentration-sensitive

• UV ( absorptivity conc)• DRI ( n/c conc)• EMD

Viscometry

Light scattering• Static ( Mw (n/c)2 conc)

o Low-angle

o Multi-angle

• Dynamic

IR

NMR

Less interested in what the detectors measure, per se, than in what the measurements can tell us about the analyte(s).

While Physical Detectors can be used alone, biggest advantage comes from synergistic combination of detectors.

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DRI, UV, IR, LS Response Depends On…

Solvent

Temperature

Wavelength

Chemical identity of analyte

Monomer ratio (copolymers)

DP (oligomers)

Why Use Physical Detectors?

Need “absolute” (i.e., calibrant-independent) M averages and/or MMD.

Need architectural information (LCB, SCB, conformation, topology, etc.).

Did analyte aggregate in solution?

Tacticity info.

Etc.

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Determining “Absolute” Molar Mass

Most accurate and precise method: Apply peak-position (calibrant-relative) calibration.

IF calibrants and analytes have the same architecture ANDchemical repeat unit.

Otherwise, can perform universal calibration using on-line viscometer and concentration-sensitive detection (e.g, DRI, UV).

Use on-line static light scattering detection (LALS, MALS), in combination with concentration-sensitive detection (e.g., DRI, UV).

Absolute M by Static Light Scattering (SLS)

Fundamental equation of SLS is the Rayleigh-Gans-Debyeapproximation:

where

and

AN

cnnK

40

220

2* 4

...2

sin3

161

)(

1 22,2

2

zGRP

R(): Excess Rayleigh scattering ratio

Mw: Weight-average molar mass

c: Concentration

A2: Second virial coefficient of solution

n0: RI of solvent at experimental temperature &wavelength

n/c: Specific refractive index increment ofsolution

0: Vacuum wavelength of incident radiation

NA: Avogadro’s cell phone number

P(): Particle scattering factor

: Angle of observation

RG,z: z-average radius of gyration

: Wavelength of radiation in medium ( 0/n0)

)(21)()(

2*

PcMAPMcK

Rww

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Obtaining Mw From a Dual-Detector System: SEC/SLS/DRI

If , then

We thus recognize that, from an SEC experiment with on-line DRI and static LS detection, we can obtain the molar mass at each slice by taking the ratio of the static LS and DRI signals at each slice.

signal

signalw DRI

SLS

c

RM

)(wM

cK

R

*

)(

Obtaining Mw From a Dual-Detector System: SEC/SLS/DRI

At each elution slice i, we can take the ratio of the signals from these two detectors, SLS and DRI (after correcting for interdetector delay or split ratio, as appropriate), to obtain the weight-average molar mass Mw as a function of elution volume and, consequently, obtain the molar mass distribution.

Retention Volume

Det

ecto

r R

espo

nse SLS

DRI

slice i

iwi

i

isignal

isignal Mc

R

DRI

SLS,

,

, )(

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Molar Mass Averages from SEC/SLS/DRI

Using static LS and DRI as detection methods in SEC, we can measure Mw,i, the molar mass at each slice i eluting from the SEC column(s).

If ci is the concentration of species of molar mass Mw,i eluting in slice i, then we can calculate the various molar mass averages of the polymer sample using the Meyerhoff equation

We can also combine the data from the concentration-sensitiveand static LS detectors to obtain the molar mass distribution.

i

xiwi

i

xiwi

Mc

McM

1,

,

when x = 0, = n; x = 1, = w; x = 2, = z

Static LS:A Molar-Mass-Sensitive Detector

As noted, the signal from a static LS detector is proportional to molar mass:

R() Mw

One application of this feature is the detection of polymeric aggregates in solution.

When polymers aggregate (for whatever reason), the molar mass of the aggregate will be greater than the molar mass of an individual polymer chain.

Therefore, the static LS signal from the aggregate will be greater than the signal produced by the unaggregated polymer.

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SLS Sensitivity to Aggregation

AUX: DRI

Aggregation of Pullulan in DMAc/0.5% LiCl, 80 oC

Striegel & Timpa, in Strategies in Size-ExclusionChromatography, Potschka & Dubin, eds. (1996) p. 366

The aggregation of pullulan in solution is easily observed with static LS. On the other hand, there is no indication of aggregation from the

refractometer signal.

Dynamic Light Scattering (DLS)

a.k.a. Quasi-Elastic LS (QELS) &Photon Correlation Spectroscopy (PCS)

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Static vs. Dynamic LS

In static light scattering (SLS), we measure the time-averaged fluctuations of the scattered light.

In dynamic light scattering (DLS, or QELS), we measure the time-dependent fluctuations of the scattered light.

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Dynamic Light Scattering (DLS)

From the measured time-dependent fluctuations in scattered light, we determine the translational diffusion coefficient, D, of the analyte.

Using D, we can calculate RH, the hydrodynamic or Stokes radius of the analyte:

Can think of RH as the radius of a homogeneous hard sphere that has the same translational diffusion coefficient as the analyte.

D

TkR B

H06

kB: Boltzmann’s constant

T: Absolute temperature of the experiment

0: Solvent viscosity at the experimental T

4.5 5.0 5.5 6.0 6.5 7.0 7.5

0.0

0.5

1.0

90o S

LS

(n

orm

aliz

ed)

Retention volume (mL)

1.0 mL min-1

PS 800K, Mw/M

n = 1.03

THF, 35 oC

1

10

100

Hyd

rod

ynam

ic radiu

s, RH

(nm

)

SEC/DLS of Narrow PS Standard

Adapted from Striegel, J. Chromatogr. A, 1359 (2014) 147

Predicted values based on RH vs. Mrelationship for PS in THF @ 25oC. SeeWernert et al., Anal. Chem. 82 (2010) 2668

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Viscometry

On-Line ViscometersTwo basic types: Single-Capillary and Differential

J Lesec, in Encyclopedia of Chromatography,2nd ed., J Cazes, Ed. (2005)

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On-Line ViscometersTwo basic types: Single-Capillary and Differential

J Lesec, in Encyclopedia of Chromatography,2nd ed., J Cazes, Ed. (2005)

Viscometry: General Principles For laminar flow within a cylinder, driven by constant pressure

gradient, the pressure drop P across a capillary is related to the length L and radius r of the tube, as well as to the viscosity of the solution flowing through the tube and to the volumetric flow rate Qthrough the tube. Whew!

The relation is given by Poiseuille’s Equation (a.k.a. the Hagen-Poiseuille Equation):

Because L, r, and Q are usually known quantities in an SEC experiment, can be directly related to P. The latter is measured using a differential pressure transducer, which measures the difference in pressure between the inlet and outlet of the cylinder.

4

8

r

QLP

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Single-Capillary Viscometers

Differential pressure transducer

measures pressure drop across tube.

At constant flow rate Q, the viscosity ηi of each slice i eluting from the SEC columns is calculated using Poiseuille’s law.

The specific viscosity of the solution, sp, is then calculated via

Pi: Pressure drop along capillary for slice i. Vi: Voltage signal corresponding to pressure drop of slice i

P0: Average solvent baseline value of pressure. V0: Average solvent baseline value of voltage

0

0

0

0

00

0 1V

VV

P

PP iisp

Single-Capillary Viscometers

REM: The intrinsic viscosity [η] is defined as

We recognize [η] as the ratio of the signal from the viscometer, which gives us ηsp, to the signal from the differential refractometer, which gives us c:

csp

c

0lim][

][

cDRI

VISC sp

signal

signal

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Obtaining [η] From a Dual-Detector System: SEC/VISC/DRI

At each elution slice i, we can take the ratio of the signals from these two detectors, VISC and DRI (after correcting for interdetector delay or split ratio, as appropriate), to obtain the intrinsic viscosity [η] as a function of elution volume and, consequently, as a function of molar mass M.

As we will see later, this [η] vs. M relationship can be quite informative.

Retention Volume

Det

ecto

r R

espo

nse VISC

DRI

slice i

ii

isp

isignal

isignal

cDRI

VISC][,

,

,

IR and NMRas

Physical Detectors

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IR as a Physical Detector: SCB of Polyolefins Monitor IR absorbance of CH3 and

combined CH2CH3 groups in polyolefins.

TCB, ODCB, and perchloroethylene are transparent in IR region of interest.

Ratio of absorbances corresponds to amount of SCB, expressed as methyl chain ends per one thousand total carbon atoms, or CH3/1000C.

For high levels of SCB, or for SCB in propylene-ethylene copolymers, calibrate using PP and PE homopolymers.

Monrabal, Sancho-Tello, LCGC Asia Pacific, September 2009

NMR as a Physical Detector:Tacticity Distribution of PMMAs

Ute et al., Macromol. Chem. Phys. 202 (2001) 3081

For more info on SEC-NMR, seetalk by Wolf Hiller on Thursday

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Obtaining Architectural Information(LCB & Conformation)

byCombining Physical Detectors

RL Whistler, in Industrial Gums, 3rd ed., RL Whistler & JN BeMiller, Eds. (1993)

Two polymers, one linear and one branched,composed of the same chemical repeat units and having the same molar

mass as each other, will occupy different hydrodynamic volumes in solutionat identical solvent/temperature conditions.

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Conformation & Mark-Houwink Plotsof Linear vs. Branched Polymers

Lo

g R

adiu

s o

f G

yrat

ion

, RG

Log Molar Mass, M

Linear

Branched

For any given M > Mx,

RG,Linear

> RG,Branched

Mx

Lo

g In

trin

sic

Vis

cosi

ty, []

Log Molar Mass, M

Linear

Branched

For any given M > Mx,

[]Linear

> []Branched

Mx

Conformation & Mark-Houwink PlotsPolymer Architecture

0.33 0.44

0.44 < 0.5

RG ~ M

Lo

g R

ad

ius

of

Gyr

atio

n, R

G

Log Molar Mass, M

0.5 < 0.6

[] ~ Ma

Lo

g In

trin

sic

Vis

cosi

ty, []

)

Log Molar Mass, M

0.5 < a 0.8

0.33 < a 0.5

0 a 0.33

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

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Relation Between Slopes of Conformation & Mark-HouwinkPlots and Polymer Architecture/Conformation

Architecture/Conformation a

Rigid rod 1 2

Linear random coil (good) 0.5 < 0.6 0.5 < a 0.8

Linear random coil () 0.5 0.5

Random branching (good) 0.44 < 0.5 0.33 < a 0.5

Random branching () 0.44 0.33

Hard sphere 0.33 0

“good” and “” correspond to values at good and theta solvent/temperature conditions, respectively

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

Polymer Conformation & Dimensionless Size

Radius of Gyration (from MALS)

31

10

3

AN

MR

i

cmiG Rrn

R 2

1

1

.

Viscometric Radius (from MALS+VISC)

Random coil of homopolymers

Hard sphere ≈ 1.3

Rigid rod0.3 – 0.4More extended

structure

More compact structure

ri: location individual atom or group of atoms

Rcm: location of polymer center of mass

n: degree of polymerization

[]: intrinsic viscosity

M: molar mass

GR

R

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Dimensionless Size & Polymeric Architecture

Architecture RG,z/RH,z

Homogeneous hard sphere 0.778

Gaussian “soft” sphere(e.g., dendrimers generation ≥ 10)

0.977

Monodisperse linear random coil-conditionsGood conditions

1.5041.78

Polydisperse linear random coil-conditionsGood conditions

1.732.05

Regular stars with uniform arm length-conditions, f = 4Good conditions, f » 1

1.3331.079

Regular stars with polydisperse arm length-conditions, f = 4Good conditions, f » 1

1.5341.225

Burchard, Adv. Polym. Sci. 1999, 143, 113

Obtain RG,z from MALS

Obtain RH,z from DLS

Combined “dimensionless ratio” is additional info not provided by either detector, individually (i.e., detector synergism).

SEC/MALS/DLS/DRI: as f(ret. vol.)

4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0

DR

I re

spo

nse

Retention volume (mL)

0.0

0.5

1.0

1.5

2.0

RG /R

H

PS1.7M, Mw/M

n = 1.10

THF, 35 oC1 mL min-1

AM Striegel, unpublished data

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Final Thoughts There is virtually no limit to how many detectors can be combined and

how this may be done (destructive detectors and backpressure issues, etc. notwithstanding).

Example 1: Ludlow et al. (J. Chromatogr. A 857 (1999) 89) performed SEC/UV/1H-NMR/ESI-MS/FT-IR to analyze polymer additives (antioxidants and plasticizers).

Example 2: Rowland & Striegel (Anal. Chem. 84 (2012) 4812) performed SEC/MALS/DLS/VISC/UV/DRI to study copolymers and blends. Determined M averages, MMD, radii, chemical heterogeneity, change in conformation as a f(M), and chemical (monomeric sequence) basis for conformational changes.

So…

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WtÇ~ }x4andre.striegel@nist.gov