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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)