PSS Webinar GPC/SEC/GFC basics · GPC/SEC/GFC basics Webinar: GPC Basics PSS Polymer Standards Service GmbH presentation for January 20, 2016 Dr. J. Preis1 and Dr. D. Lohmann2 1 Polymer

GPC/SEC/GFC basics

Webinar: GPC Basics

PSS Polymer Standards Service GmbH presentation for

January 20, 2016

Dr. J. Preis1 and Dr. D. Lohmann2

1 Polymer Standards Service (PSS) GmbH; Mainz, Germany; 2 Polymer Standards Service (PSS) USA; Amherst, MA; USA

About PSS

Provides instruments and turn-key systems for the efficient and

comprehensive characterization of macromolecules.

Manufactures high-resolution GPC/SEC columns and

develops new stationary phases.

Develops compliant software for real-time data

acquisition and powerful and easy-to-use data evaluation.

Manufactures reference materials and validation kits.

Offers contract analysis, method development, validation services, training

courses and webinars.

PSS Headquarter with production facility and R&D center is located in Mainz,

Germany.

Webinar: GPC Basics Page 2

Outline

• Introduction

• Fundamentals

• Principle of separation

• Hydrodynamic volume and intrinsic viscosity

• Molar mass average values Mw and Mn

• Calibration and resolution

• Method development

• Detection

• Concentration detectors

• Molar mass sensitive detectors


Introduction


Macromolecules are everywhere

Automotive

Construction

Cosmetics

Feed & Food

Packaging

Paper & Wood

Pharma

Introduction


Synthesis of macromolecules

One or more different monomers form chains

Molar mass distribution (MMD)

Chemical composition distribution (CCD)

Molecular architecture distribution (MAD)

Structural type distribution (STD)

Functionality type distribution (FTD)

Introduction


Solution Molar mass distr.(MMD) Molecular architecture distr. (MAD)

Separation GPC/SEC GPC/SEC

Detection RI, UV, ELSD, MS,

Viscometer/LS + RI/UV

Viscometer/LS + RI/UV


Polystyrene

SDV, 3μm,

3 x 100, 3 x 1000,

THF, 0.25ml/min

W (

log M

)

molar mass

Question:

• What is the molar mass of

such a polymer?

• How can it be quantified?

Molar mass average values

Molar mass average values

How can we describe a polymer chain distibution

What is the molar mass of a polymer?

What are molar mass average values?


> 1

Number average molar mass Weight average molar mass

Polydispersity index

Molar mass distribution (MWD)

GPC offers the opportunity to detect the whole molar mass or molecular

weight distribution (MWD) of a polymer

MWD tells you the “true story” about the polymer

Mn and Mw are only single values


0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

2.25

2.50

Molar mass [Da]

1*105

5*105

1*106

W(log

M)

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Proj ekt : Kostenstelle :Datum : Zeichen :

W:\GPC_DAT\Vorab_09.LDXDienstag 09/07/21 10:57:13

PNBMA UV 230nm

PNBMA RI

0

10

20

30

40

50

60

70

80

90

100

molar mass [Da]

1*102

1*103

1*104

1*105

norm

. W

(log M

)

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Example: MWD of Polymers

Low molar mass tailing

bimodal distribution

Difference between GPC/SEC and LAC

GPC/SEC

GPC/SEC is a pore size

limited, diffusion controlled,

partition chromatography

A pure GPC/SEC

mechanism has no

interaction with the matrix

GPC/SEC needs a polarity

balanced system.


LAC

• Interaction chromatography

(LAC) uses the

differences in the chemical

(polarity) or

structural nature of the

sample for the

separation

Polymer Interaction I

Homopolymer A


Homopolymer B

Adsorption of homopolymer

B on the matrix

Polymer Interaction II



GPC/SEC

different size = different hydrodynamic radius different color = different chemistry

GPC/SEC Column

Separation by Size

How does GPC/SEC work?

GPC = Gel permeation chromatography

SEC = Size exclusion chromatography

GPC/SEC Basics

GPC/SEC Separation is based on the size of a molecule in solution

Different molar mass ->

Different size/color =

different hydrodynamic

radius

Detector measures a

concentration profile


GPC/SEC Basics

Primary results GPC/SEC:

Chromatogram with peaks at different

elution volume, with different shapes

and intensities

From Chromatogram to the Molar

Mass Distribution (MMD):

Required:

Molar mass at elution volume

- Calibration curve (reference

materials required)

GPC/SEC is a so called „relative

method“ for molar mass

determination

5

10

15

20

25

30

35

40

45

Elution v olume [ml]

5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0

RI S

igna

l [mV

]

PSS W

inGPC

Unity,

Build

3523

, LAB

-SW1

, Insta

nce #

1

? ? ? ?


Calibration standards


pre-weighted kits

or

single reference standards

Important are:

Narrow standards

(MWD)

Molar mass average

value information

Mp information

handling information

Measurement

conditions

10 11 12 13 14 15 16 0,000

0,005

0,010

0,015

0,020

0,025

0,030

Elugram

Elution Volume [ml]

10 12 14 16 18 20 10 2

10 3

10 4

10 5

10 6

Calibration Curve

Mo

lma

ss [D

a]

Elution Volume [ml]

Introduction to GPC/SEC


Elugram and calibration curve leads to

molar mass information

GPC/SEC is a so called relative method

Molar mass information based on

calibration

PSS live Webinar: March 9, 2016

Hydrodynamic volume


The separation parameter in GPC/SEC is not the molar

mass but the hydrodynamic volume Vh of a polymer

Vh is a function of the molar mass M and the intrinsic

viscosity [η]

The most important equations are:

Vh = [η] x M

[η] = K x Mα

polymer size in different eluents

same polstyrene in:

methanol cyclo-

hexan

THF chloroform

elution volume for different

polymers in same eluent

polyisoprene

M = 15000

polystyrene

M = 20000

PMMA

M = 24000

Vh (PI) = Vh (PSt) = Vh (PMMA)

but not the molar mass!!

Hydrodynamic volume Polymer - Solvent

Interactions


GPC/SEC system

Required Items


Columns are the heart of the system!

Without sufficient separation only limited information

(even when intelligent detection is used).

Isocratic pump

Injection system

One or more detectors

(depending on

application)

Calibration standards, reference materials, validation standards

GPC/SEC data

acquisition

and evaluation

software

GPC/SEC


How to select a GPC/SEC column?

Allow interaction free

chromatography: choose the right

stationary phase and method

Balanced polarities for interaction

free chromatography

PSS Magic Triangle

PSS Column Selector:

www.psscolumnselector.com

PSS live Webinar:

February 17, 2016

Optimizing a method I


elution v olume [ml]

6 7 8 9 10 11 12 13 14 15 16

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Dextran in pure Water

SUPREMA 1000Å; 10µm, 8x300mm

Flow rate: 1ml/min; RT

Conc.: 1[g/l]

Quality issue?

molar mass: 5000[g/mol] - 277.000[g/mol]

Detector: RI [V]

Interpretation of the

elugrams: What does this really

means?

Possible reasons for a

multimodale distributions

Optimizing a method II


elution v olumen [ml]

6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0 12.5

PS

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Dextran in aqueous solution

SUPREMA 1000Å; 10µm, 8x300mm

Flow rate: 1ml/min; RT

Conc.: 1[g/l]

Additive:NaN3; 5 [g/l]molar mass: 10000[g/mol] - 500.000[g/mol]

Detector: RI [V] no associates !!

Salt effect in the

aqueous

SEC/GPC

Bimodal

distribution is not

nescesarrily a

quality issue of the

column


Questions

SEC Method development

Webinar: GPC Basics

0.000

0.001

0.002

0.003

0.004

0.005

0.006

0.007

0.008

0.009

0.010

Elution Volume [ml]

8 9 10 11 12 13 14 15 16 17 18

Dete

cto

r R

esponse [

V]

flow rate: 0,25ml/min

Supre

ma 1

0µ

m,

100Å

Supre

ma 5

µm

, 100Å

Suprema 5µm, 100Å,100Å

Sample: dxt T1

Influence of particle size, temperature and separation distance

Particle size, separation distance and temperature increase the resolution by > 100%

Smaller particle leads to higher back pressure (shear degradation)

Page 25

Calibration curves

4 5 6 7 8 9 10 11 12

102

103

104

105

106

107 50Å

100Å

500Å

1000Å

1E4Å

1E5Å

1E6Å

1E7Å

SDVM

ola

r M

ass [D

]

Elution Volume [ml]


• Porosity defines the separation range

Resolution

Influence of

the porosity

Pore type Advantage Disadvantage

Single porosity efficient, molar mass optimized, Viscous fingering

Linear/mixed

porosity

fast, universal, cost effective Poor resolution /

(Efficiency)


Resolution vs time


Set of columns:

+ High resolution

+ Wide separation range

Eluent/time consumption

Linear column:

+ Wide separation range

+ Short analysis time

Lower resolution

Combination of columns with single porosities vs. linear column

40

Column Characteristics

Column Type/Dimensions

Task: Find the column that meets your application needs

Preparative HighSpeed Analytical Semi-micro

Typical Dimensions

[mm]

ID: 20 mm

Length: 300

ID: 20 mm

Length: 50

ID: 8 mm

Length: 300

ID: 4.6 mm

Length: 250

Flow-rate [mL/min] 3 - 10 3 - 10 0.5 – 1 0.2 - 0.5

Time consumption

per column [min]

12.5 2 12.5 10

Eluent consumption

per column [mL]

78.1

(6.25 mL/min)

12.5

(6.25 mL/min)

12.5

(1 mL/min)

3.5

(0.3 mL/min)

Maximum loading per

column*

10 mg/mL,

Up to 1000 µL

2 mg/ml,

20 µL

2 mg/ml,

20 µL

2 mg/ml,

5 µL


* Depends largely on molar mass and polydispersity, loading maximized (not separation)

Column Characteristics Chemistry: Column Material

Polymer packing Inorganic packing

Polarity Non Medium Polar Non Medium Polar

Chemistry St-DVB Acrylic,

Polyester

OH-acrylic SiO2-C18 SiO2-diol SiO2

Solvents THF, TCM,

Toluene

DMF, NMP,

DMAc,

DMSO, (all)

Water

(pH 2 to 13)

Aqueous

(pH < 9)

organic/DMAc

Examples SDV,

POLEFIN

GRAM SUPREMA,

NOVEMA

Max

PROTEEMA,

POLARSIL,

PFG

Samples PS, PMMA

PVC, PC,

Resins, PE,

PP, etc.

PU, Starch,

Cellulose,

Polyimide

etc.

PEG/PEO,

Pullulan,

Dextran,

PAA, etc.

Proteins,

Petides, PLA,

Polyester,

POM, etc.


GPC/SEC


How to select a detection method?

Always required: Concentration detector (Refractive index RI, UV/DAD,

evaporative light scattering ELSD)

Determination of molar mass distribution and averages Mw, Mn, PDI, …

Calibration with molar mass standards,

broad calibration, cumulative calibration, universal calibration:

- True molar masses for chemically identical substances

- Ideal for QC

- Good reproducibility

Typical Detection Techniques

Signal intensity

concentration detector: X = 0 UV: kSample = extinction coefficient

light scattering detector: X = 1 RI: kSample = refractive index increment dn/dc

viscometer: X = MH-

mass spec, osmometer: X = -1

I K k c MD Sample Samplei

x( )

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

Elution Volume [ml]

13 14 15 16 17 18 19 20

Vol

tage

[V]

PS

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inG

PC

sc

ien

tifi

c

I (V)LS

I (V)visco

I (V)osm

I (V)conc

Page 32 Webinar: GPC Basics

Page 32

Typical molar mass sensitive detectors: light scattering, viscometer

Molar Mass Sensitive Detection

and Applications

Page 33

4 different molar masses: 2 570 000 Da, 250 000 Da, 17 800 Da, 1620 Da

Concentration: 0.5 mg/mL, 1 mg/mL, 1 mg/mL, 1 mg/mL

RI reflects injected mass; Visco/LS injected mass and molar mass

Webinar: GPC Basics


column viscometer RI

visco [ ]·c RI c

visco/RI [ ] + universal calibration

curve log([ ]·M)

column LS RI

LS (dn/dc)2 ·M·c RI c

LS/RI (dn/dc)2 ·M

M

M

Molar Mass Sensitive Detection

and Applications

PSS Webinar Series 2013


Light Scattering

zylindrische Meßzelle

Lichtwellenleiter

(Eluat ein/aus: unter/über Ebene)

(Winkelpos ition)

LASER

cylindrical cell

(solvent in/out:

under/above

plane)

optical fibre

(angle position)

Cell design 7 angle SLD7000


Application Example Viscometer:

In combination with RI used to measure the intrinsic viscosity [ ]

PSS DVD1260

Viscometer bridge design :

2 signals from viscometer:

Delta pressure

Inlet pressure

Differential pressure

Inlet pressure

R2

R4

B

R1

A

R3

From Columns

Waste/concentration detector

Sample path Solvent path

Viscometer


Visco/LS

Advantages Light Scattering

- Absolute method: measures directly M at every elution volume

- No calibration standards required

Ideal for (homopolymer) high molar mass samples, and proteins where

absolute molar masses are required. Advantages Viscometry

Measures intrinsic viscosity directly

Independent of refractive index increment dn/dc

Absolut molar mass for copolymers

Branching information

Universal calibration required Ideal for homopolymers , copolymers and branched samples

PSS live Webinar : June 8 , 2016

PSS live Webinar: September 14 , 2016

Summary

GPC/SEC is a well established method for polymer characterization

GPC/SEC is relative method, calibration is required

True molar masses only if polymer and calibration standards are the same polymer type

Mw, Mn and Mp and any other molar mass average value and PDI can be determined

Mechanism is purely entropic, no interactions

Separation according to the size of the molecule, Vh not the molar mass

Porosity and particle size of the columns have to be chosen according to the separation task

GPC/SEC in combination with hyphenated coupling techniques provide deeper inside into polymer properties (branching, composition or absolute molar mass)


PSS GPC/SEC Training Cource (Theory and Praxis) in Mainz/Germany: September 19-20 , 2016

Thanks for your kind attention

If you have any further question please

contact

[email protected]

or

[email protected]


mailto:[email protected]






Documents

PSS Webinar GPC/SEC/GFC basics · GPC/SEC/GFC basics Webinar: GPC Basics PSS Polymer Standards Service GmbH presentation for January 20, 2016 Dr. J. Preis1 and Dr. D. Lohmann2 1 Polymer