Addressing the challenge of analysing low molecular weight samples by multi detection GPC

Bert Postma Business Support Manager - Separations

Introduction

› Triple detection GPC/SEC using RI, viscometer and light scattering detection is now an established technique used by hundreds of laboratories world-wide in all areas of polymer and protein research.

› However, that doesn't mean it is without challenges, and one of the most common is the issue of the analysis of low molecular weight samples.

› This presentation will give some examples of challenging low molecular weight applications and the technology that lies behind their successful analysis.

Why are low molecular weight samples a challenge?

› Concentration detector response (e.g. RI or UV) is proportional to concentration

› Light scattering or viscometer detectors are sensitive to concentration and molecular weight or molecular viscosity

› Detector ‘sensitivity’ therefore decreases across a sample elution profile from high to low molecular weight

RI Chromatogram PS10-1 RI ps1-1 RI ps11-1 RI ps12-1 RI ps2-1 RI ps3-1 RI ps4-1 RI ps5-1 RI ps6-1 RI ps7-1 RI ps8-1 RI ps9-1 RI

8,0 10,2 12,4 14,7 16,9 19,1 21,3 23,6 25,8 28,0 -20

-2

15

33

50

68

86

103

121

138

Retention Volume (mL)

Res

pons

e (m

V)

PolyCal standards

Viscosity Chromatogram PS10-1 DP ps1-1 DP ps11-1 DP ps12-1 DP ps2-1 DP ps3-1 DP ps4-1 DP ps5-1 DP ps6-1 DP ps7-1 DP ps8-1 DP ps9-1 DP

8,0 10,2 12,4 14,7 16,9 19,1 21,3 23,6 25,8 28,0 -70

-29

12

52

93

134

175

216

257

298

Retention Volume (mL)

Res

pons

e (m

V)

PolyCal standards

Sensitivity of RI Detector for Low Mw

-28,6

63,2

-20,0

-10,0

0,0

10,0

20,0

30,0

40,0

50,0

PS

9 K

(55)

/ M

etho

d: P

S-0

000.

vcm

6,0

14,2

Retention Volume (mL)

7,0

8,0

9,0

10,0

11,0

12,0

13,0

Overlay Plot: Refractive Index (mV) Vs. Retention Volume (mL) Method: PS-0000.vcm

8,4

4

Low Mw PS standards: 100 ul, 1 mg/ml, 18K, 9K, 8K, 5K, 2K, 1K, 374, 162

Sensitivity of Viscometer for Low Mw

-39,0

-18,6

-36,0

-33,0

-30,0

-27,0

-24,0

-21,0

PS

9 K

(55)

/ M

etho

d: P

S-0

000.

vcm

6,0

14,0

Retention Volume (mL)

7,0

8,0

9,0

10,0

11,0

12,0

13,0

Overlay Plot: Viscometer DP (mV) Vs. Retention Volume (mL) Method: PS-0000.vcm

Low Mw PS standards: 100 ul, 1 mg/ml, 18K, 9K, 8K, 5K, 2K, 1K, 374, 162

Three different examples

› Heparin Natural low molecular weight material with broad

distribution

› PE waxes

High temperature GPC application

› PEG/PLA complexes Low Mw Branching

Heparin

Low Molecular Weight Heparin (LMWH)

› Important and widely prescribed anticoagulant

› Naturally occurring highly sulphated glycosaminoglycan

› LMWH derived from Unfractionated Heparin (UFH) UF sourced from the mucosal tissue of porcine intestines. Manufacturers use different de-polymerization processes

› LMWH has an expected Mw range 3kDa – 7kDa

› LMWH has a number of advantages over UFH

Better bioavailability More predictable dose response Improved plasma half life

› Three samples compared using TDA Celexane, Fragmin, Tinzaparin

› Despite low molecular weight and low IV the data is good › New high resolution columns allow degrees of polymerisation to be

observed.

TDA Chromatogram

› Weight fractions show significant differences between three LMWH › Celexane has more material with a lower degree of polymerisation.

Heparin – Weight fraction distribution

› Structural comparisons between samples are made using the Mark-Houwink relationship

› All samples show similar structure

Heparin – Structural comparison

Log [η] = Log K + aLogM Plot

› How is it actually called? › Mark-Houwink plot

Herman Mark (Austrian - American) Roelof Houwink (Netherlands) Ichiro Sakurada (Japan) Werner Kuhn (Switzerland)

› Die Beziehung zwischen [η] und M wurde von Hermann Staudinger empirisch bei Messungen an Cellulose und Cellulosederivaten entdeckt. Werner Kuhn begründete sie durch seine Beschreibung des statistischen Knäuels. Hermann Mark formulierte sie erstmals in dieser Form, und Roelof Houwink bestätigte ihre Gültigkeit durch Messungen verschiedener Polymere in unterschiedlichen Lösemitteln. Sakurada published it.

› Viscotek finally used it

Log [η] = Log K + aLogM Log [Rg] = Log k + αLogM

Conformation Plotnbs706-1 Rg LSnbs706-2 Rg LSps250k-1 Rg LSps250k-2 Rg LSpsrr1-1 Rg LSpsrr1-2 Rg LS

5,00 5,25 5,50 5,75 6,00 1,05

1,20

1,35

1,50

1,65

Log(Molecular Weight)

Log[

Rad

ius

of G

yrat

ion]

0 < a < 2 0.3 < α < 1

Mark-Houwink Plot nbs706-1 IV LS nbs706-2 IV LS ps250k-1 IV LS ps250k-2 IV LS psrr1-1 IV LS psrr1-2 IV LS

5,00 5,25 5,50 5,75 6,00 -4,00

-2,03

-0,05

1,93

3,90

Log(Molecular Weight)

-1

Log[

Intri

nsic

Vis

cosi

ty]

The Analysis of Low Molecular Weight PE Wax by Advanced Triple Detection HT-GPC

Applications

› Sample 1: Polymer Additives for candles flow hardness strength

• Used as partial or total replacement • Described as a hyper branched polyolefin

› Sample 2: Synthetic Wax

Similar structure to paraffin wax, • higher melting point, • hardness • molecular weight.

Used in hot melt adhesives.

Measurement Conditions

› HT GPC at 150°C › TCB + 500ppm BHT › 3 mixed bed HT GPC columns › LALS-RI-Viscometer detection

Triple Detection Chromatogram for Sample 1 Hyper branched Polyolefin

Ref

ract

ive

Inde

x (m

V)

942

943

944

945

946

947

948

949

950

Rig

ht A

ngle

Lig

ht S

catte

ring

(mV

)

130

140

150

160

170

180V

isco

met

er -

DP

(mV

)

8 9 10 11 12 13 14 15 16 17 18 19 20 21 22Retention Volume (mL)

2010-12-03_00;42;31_Vybaw_253_01-0002.vdx : Refractive Index (mV)2010-12-03_00;42;31_Vybaw_253_01-0002.vdx : Right Angle Light Scattering (mV)2010-12-03_00;42;31_Vybaw_253_01-0002.vdx : Viscometer - DP (mV)

Triple Detection Chromatogram Sample 2 Synthetic Wax

Re

fra

ctiv

e I

nd

ex

(mv)

941

942

943

944

945

946

947

948

949

Rig

ht

An

gle

Lig

ht

Sca

tte

ring

(m

v)

-200

-100

0

100

200

Vis

com

ete

r -

DP

(m

v)

10 11 12 13 14 15 16 17 18 19 20 21 22 23Retention Volume (mL)

2010-12-02_23;48;44_Cue_PauaFlin_C80_02-0000.vdx : Refractive Index (mv)2010-12-02_23;48;44_Cue_PauaFlin_C80_02-0000.vdx : Right Angle Light Scattering (mv)2010-12-02_23;48;44_Cue_PauaFlin_C80_02-0000.vdx : Viscometer - DP (mv)

0.1

0.2

1

2

10 R

h (n

m)

0

1

2

3

4

5 W

F / d

Log

MW

100 200 300 400 500 1000 2000 3000 Molecular Weight (Da)

Rh (nm) WF / dLog MW

Overlay of the MWD and Rh-plot for ‘Sample 2’

Structural confirmation of synthesised PEG/PLA Copolymer using multiple detection SEC

Effect of PLA structure on molecular parameters

D-Lactide

PDLA

L-Lactide

PLLA

Meso – Lactide

PDLLA

Triple Detection SEC Data

Sample Id Mw Mn Mw/Mn IV Rh PEG 13,062 12,839 1.02 0.158 3.2 PEG-PLA L 36,728 34,300 1.07 0.342 5.8 PEG-PLA 4 61,177 44,846 1.36 0.345 6.8 PEG-PLA 8 74,286 62,206 1.19 0.259 6.6

0

50

100

150

200

250

300

Ref

ract

ive

Inde

x (m

v)

-15

-10

-5

0

5

10

15

20

Rig

ht A

ngle

Lig

ht S

catte

ring

(mv)

-15

-10

-5

0

5

10

15

20

Low

Ang

le L

ight

Sca

tterin

g (m

v)

-300

-250

-200

-150

-100

-50

0

50

Visc

omet

er -

DP

(mv)

10 15 20 25 30 35 40 45Retention Volume (mL)

2012-06-26_05;04;14_PDLA_02-0004.vdx : Refractive Index (mv)2012-06-26_05;04;14_PDLA_02-0004.vdx : Right Angle Light Scattering (mv)2012-06-26_05;04;14_PDLA_02-0004.vdx : Low Angle Light Scattering (mv)2012-06-26_05;04;14_PDLA_02-0004.vdx : Viscometer - DP (mv)

Structure of PEG Homopolymer

0.1

0.2

0.3

0.4

1

Intri

nsic

Vis

cosi

ty (d

L/g)

4000 5000 60004

104

2x104

3x104

4x104

5x104

6x105

10Molecular Weight (Da)

2012-01-24_15;57;38_UR1_03 opt-0000.vdx : Intrinsic Viscosity (dL/g)2012-01-24_16;33;53_UR_2_01 BN-0001.vdx : Intrinsic Viscosity (dL/g)2012-01-24_18;22;36_UR_3__01 BN-0000.vdx : Intrinsic Viscosity (dL/g)

Confirmation of PEG Branched state

UR2 4.2 Arms UR3 8.2 Arms

Structure of PEG-PLA Copolymer

Quantitative Results - Branching

UR5 4.8 Arms UR6 8.5 Arms

The key to successful Low MW analysis

› Good chromatography › Optimised conditions (dn/dc) › Stable detector baselines › High detector sensitivity (s/n)