summary of results from sec-mals analysis 1 copyright of wyatt technology corporation...
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1Copyright of Wyatt Technology Corporation
Summary of Results from SEC-MALS Analysis
Polysaccharides Characterized by SEC-MALS
Presented for
By Shawn Cao, Ph.D.
Wyatt Technology Corporation
6300 Hollister Avenue
Santa Barbara, CA 93117
Phone: (805) 681-9009
URL: http://www.wyatt.com e-mail: [email protected]
2Copyright of Wyatt Technology Corporation
Summary of Results from SEC-MALS Analysis
SEC-MALS System
SEC system: Agilent HP1100 HPLC System
MALS detector: DAWN EOS from Wyatt
Concentration detector: Optilab rEX DRI from Wyatt
3Copyright of Wyatt Technology Corporation
Summary of Results from SEC-MALS Analysis
Chromatograms & Results forPA Softwood
4Copyright of Wyatt Technology Corporation
Summary of Results from SEC-MALS Analysis
Chromatograms: PA Softwood
-0.1
0.0
0.1
0.2
0.3
0.4
0 10 20 30
LS #
11, A
UX
1
Time (min)
11
Peak ID - Weyer_soft_1mgmL_b_01LS #11AUX1
LSRI
5Copyright of Wyatt Technology Corporation
Summary of Results from SEC-MALS Analysis
Debye Plot of One Slice of the Above Chromatogram
90° & AUX detector
Peak, Slice : 1, 853 Time : 14.217 min Fit degree : 2 Conc. : (3.036 ± 0.006)e-5 g/mL Mw : (7.915 ± 0.644)e+5 g/mol Radius : 97.0 ± 9.3 nm
-48.00x10
-31.20x10
-31.60x10
-32.00x10
-32.40x10
0.0 0.2 0.4 0.6 0.8 1.0
sqrt[
K*c
/R(th
eta)
]
sin²(theta/2)
Debye Plot - Weyer_soft_1mgmL_b_01
6Copyright of Wyatt Technology Corporation
Summary of Results from SEC-MALS Analysis
Distribution: MM vs. Elution Time
41.0x10
51.0x10
61.0x10
71.0x10
10.0 12.0 14.0 16.0 18.0 20.0
Mol
ar M
ass
(g/m
ol)
Time (min)
Molar Mass vs. Time Weyer_soft_1mgmL_b_01
Mw = 7.7 x 105 (± 10%) PDI = 2.9 (± 31%)
7Copyright of Wyatt Technology Corporation
Summary of Results from SEC-MALS Analysis
Distribution: Cumulative MM Distribution
0.0
0.2
0.4
0.6
0.8
1.0
1.0x10 4 1.0x10 5 1.0x10 6 1.0x10 7
Cum
ulat
ive
Wei
ght F
ract
ion
W
Molar Mass (g/mol)
Cumulative Molar Mass Weyer_soft_1mgmL_b_01
8Copyright of Wyatt Technology Corporation
Summary of Results from SEC-MALS Analysis
Distribution: RMS vs. Elution Time
10.0
100.0
1000.0
10.0 12.0 14.0 16.0 18.0 20.0
R.M
.S. R
adiu
s (n
m)
Time (min)
RMS Radius vs. Time Weyer_soft_1mgmL_b_01
9Copyright of Wyatt Technology Corporation
Summary of Results from SEC-MALS Analysis
Conformational plot: Log(RMS) vs. Log(MM)
10.0
100.0
1000.0
1.0x10 4 1.0x10 5 1.0x10 6 1.0x10 7
R.M
.S. R
adiu
s (n
m)
Molar Mass (g/mol)
RMS Radius vs. Molar Mass Weyer_soft_1mgmL_b_01
Slope = 0.43
10Copyright of Wyatt Technology Corporation
Summary of Results from SEC-MALS Analysis
Duplicate injection to show reproducibility
41.0x10
51.0x10
61.0x10
71.0x10
81.0x10
10.0 12.0 14.0 16.0 18.0 20.0
Mol
ar M
ass
(g/m
ol)
Time (min)
Molar Mass vs. Time Weyer_soft_1mgmL_b_01Weyer_soft_1mgmL_b_02
11Copyright of Wyatt Technology Corporation
Summary of Results from SEC-MALS Analysis
Duplicate injection to show reproducibility
0.0
0.2
0.4
0.6
0.8
1.0
1.0x104 1.0x105 1.0x106 1.0x107 1.0x108
Cum
ulat
ive
Wei
ght F
ract
ion
W
Molar Mass (g/mol)
Cumulative Molar Mass Weyer_soft_1mgmL_b_01Weyer_soft_1mgmL_b_02
12Copyright of Wyatt Technology Corporation
Summary of Results from SEC-MALS Analysis
Duplicate injection to show reproducibility
10.0
100.0
1000.0
10.0 12.0 14.0 16.0 18.0 20.0
R.M
.S. R
adiu
s (n
m)
Time (min)
RMS Radius vs. Time Weyer_soft_1mgmL_b_01Weyer_soft_1mgmL_b_02
13Copyright of Wyatt Technology Corporation
Summary of Results from SEC-MALS Analysis
Duplicate injection to show reproducibility
10.0
100.0
1000.0
1.0x104 1.0x105 1.0x106 1.0x107 1.0x108
R.M
.S. R
adiu
s (n
m)
Molar Mass (g/mol)
RMS Radius vs. Molar Mass Weyer_soft_1mgmL_b_01Weyer_soft_1mgmL_b_02
14Copyright of Wyatt Technology Corporation
Summary of Results from SEC-MALS Analysis
Chromatograms & Results for9H4F
15Copyright of Wyatt Technology Corporation
Summary of Results from SEC-MALS Analysis
Chromatograms: 9H4F
-1.2
-1.1
-1.0
-0.9
-0.8
0 10 20 30
Det
ecto
r: A
UX
1
Time (min)
0.2
0.3
0.4
0.5
0.6
0.7
Det
ecto
r: 1
1
Strip Chart - Weyer_9H4F_1mgmL_b_01
16Copyright of Wyatt Technology Corporation
Summary of Results from SEC-MALS Analysis
Debye Plot of One Slice of the Above Chromatogram
90° & AUX detector
Peak, Slice : 1, 843 Time : 14.050 min Fit degree : 2 Conc. : (2.966 ± 0.005)e-5 g/mL Mw : (1.070 ± 0.012)e+6 g/mol Radius : 113.5 ± 1.5 nm
-48.00x10
-31.00x10
-31.20x10
-31.40x10
-31.60x10
-31.80x10
0.0 0.2 0.4 0.6 0.8 1.0
sqrt[
K*c/
R(th
eta)
]
sin²(theta/2)
Debye Plot - Weyer_9H4F_1mgmL_b_01
17Copyright of Wyatt Technology Corporation
Summary of Results from SEC-MALS Analysis
Distribution: MM vs. Elution Time
51.0x10
61.0x10
71.0x10
81.0x10
91.0x10
10.0 12.0 14.0 16.0 18.0
Mol
ar M
ass
(g/m
ol)
Time (min)
Molar Mass vs. Time Weyer_9H4F_1mgmL_b_01
Mw = 3.1 x 106 (± 10%) PDI = 3.7 (± 14%)
18Copyright of Wyatt Technology Corporation
Summary of Results from SEC-MALS Analysis
Distribution: Cumulative MM Distribution
0.0
0.2
0.4
0.6
0.8
1.0
1.0x10 5 1.0x10 6 1.0x10 7 1.0x10 8 1.0x10 9
Cum
ulat
ive
Wei
ght F
ract
ion
W
Molar Mass (g/mol)
Cumulative Molar Mass Weyer_9H4F_1mgmL_b_01
19Copyright of Wyatt Technology Corporation
Summary of Results from SEC-MALS Analysis
Distribution: RMS vs. Elution Time
10.0
100.0
1000.0
10000.0
10.0 12.0 14.0 16.0 18.0
R.M
.S. R
adiu
s (n
m)
Time (min)
RMS Radius vs. Time Weyer_9H4F_1mgmL_b_01
20Copyright of Wyatt Technology Corporation
Summary of Results from SEC-MALS Analysis
Conformational plot: Log(RMS) vs. Log(MM)
10.0
100.0
1000.0
10000.0
1.0x10 5 1.0x10 6 1.0x10 7 1.0x10 8 1.0x10 9
R.M
.S. R
adiu
s (n
m)
Molar Mass (g/mol)
RMS Radius vs. Molar Mass Weyer_9H4F_1mgmL_b_01
Slope = 0.34
21Copyright of Wyatt Technology Corporation
Summary of Results from SEC-MALS Analysis
Duplicate injection to show reproducibility
51.0x10
61.0x10
71.0x10
81.0x10
91.0x10
10.0 12.0 14.0 16.0 18.0
Mol
ar M
ass
(g/m
ol)
Time (min)
Molar Mass vs. Time Weyer_9H4F_1mgmL_b_01Weyer_9H4F_1mgmL_b_02
22Copyright of Wyatt Technology Corporation
Summary of Results from SEC-MALS Analysis
Duplicate injection to show reproducibility
0.0
0.2
0.4
0.6
0.8
1.0
1.0x10 5 1.0x10 6 1.0x10 7 1.0x10 8 1.0x10 9
Cum
ulat
ive
Wei
ght F
ract
ion
W
Molar Mass (g/mol)
Cumulative Molar Mass Weyer_9H4F_1mgmL_b_01Weyer_9H4F_1mgmL_b_02
23Copyright of Wyatt Technology Corporation
Summary of Results from SEC-MALS Analysis
Duplicate injection to show reproducibility
10.0
100.0
1000.0
10000.0
10.0 12.0 14.0 16.0 18.0
R.M
.S. R
adiu
s (n
m)
Time (min)
RMS Radius vs. Time Weyer_9H4F_1mgmL_b_01Weyer_9H4F_1mgmL_b_02
24Copyright of Wyatt Technology Corporation
Summary of Results from SEC-MALS Analysis
Duplicate injection to show reproducibility
10.0
100.0
1000.0
10000.0
1.0x10 5 1.0x10 6 1.0x10 7 1.0x10 8 1.0x10 9
R.M
.S. R
adiu
s (n
m)
Molar Mass (g/mol)
RMS Radius vs. Molar Mass Weyer_9H4F_1mgmL_b_01Weyer_9H4F_1mgmL_b_02
25Copyright of Wyatt Technology Corporation
Summary of Results from SEC-MALS Analysis
Chromatograms & Results forWeyco #20
26Copyright of Wyatt Technology Corporation
Summary of Results from SEC-MALS Analysis
Chromatograms: Weyco #20
-0.7
-0.6
-0.5
-0.4
-0.3
-0.2
0 10 20 30
Det
ecto
r: A
UX1
Time (min)
0.25
0.30
0.35
0.40
0.45
Det
ecto
r: 1
1
Strip Chart - Weyer_20_1mgmL_b_03
27Copyright of Wyatt Technology Corporation
Summary of Results from SEC-MALS Analysis
Debye Plot of One Slice of the Above Chromatogram
90° & AUX detector
Peak, Slice : 1, 853 Time : 14.217 min Fit degree : 2 Conc. : (1.146 ± 0.004)e-5 g/mL Mw : (8.617 ± 0.111)e+5 g/mol Radius : 67.3 ± 2.4 nm
-31.00x10
-31.10x10
-31.20x10
-31.30x10
-31.40x10
-31.50x10
0.0 0.2 0.4 0.6 0.8 1.0
sqrt[
K*c/
R(th
eta)
]
sin²(theta/2)
Debye Plot - Weyer_20_1mgmL_b_03
28Copyright of Wyatt Technology Corporation
Summary of Results from SEC-MALS Analysis
Distribution: MM vs. Elution Time
41.0x10
51.0x10
61.0x10
71.0x10
81.0x10
10.0 12.0 14.0 16.0 18.0 20.0
Mol
ar M
ass
(g/m
ol)
Time (min)
Molar Mass vs. Time Weyer_20_1mgmL_b_03
Mw = 2.4 x 105 (± 3%) PDI = 3.0 (± 9%)
29Copyright of Wyatt Technology Corporation
Summary of Results from SEC-MALS Analysis
Distribution: Cumulative MM Distribution
0.0
0.2
0.4
0.6
0.8
1.0
1.0x10 4 1.0x10 5 1.0x10 6 1.0x10 7 1.0x10 8
Cum
ulat
ive
Wei
ght F
ract
ion
W
Molar Mass (g/mol)
Cumulative Molar Mass Weyer_20_1mgmL_b_03
30Copyright of Wyatt Technology Corporation
Summary of Results from SEC-MALS Analysis
Distribution: RMS vs. Elution Time
10.0
100.0
1000.0
10.0 12.0 14.0 16.0 18.0 20.0
R.M
.S. R
adiu
s (n
m)
Time (min)
RMS Radius vs. Time Weyer_20_1mgmL_b_03
31Copyright of Wyatt Technology Corporation
Summary of Results from SEC-MALS Analysis
Conformational plot: Log(RMS) vs. Log(MM)
10.0
100.0
1000.0
1.0x10 4 1.0x10 5 1.0x10 6 1.0x10 7 1.0x10 8
R.M
.S. R
adiu
s (n
m)
Molar Mass (g/mol)
RMS Radius vs. Molar Mass Weyer_20_1mgmL_b_03
32Copyright of Wyatt Technology Corporation
Summary of Results from SEC-MALS Analysis
Duplicate injection to show reproducibility
41.0x10
51.0x10
61.0x10
71.0x10
81.0x10
10.0 12.0 14.0 16.0 18.0 20.0
Mol
ar M
ass
(g/m
ol)
Time (min)
Molar Mass vs. Time Weyer_20_1mgmL_b_03Weyer_20_1mgmL_b_01
33Copyright of Wyatt Technology Corporation
Summary of Results from SEC-MALS Analysis
Duplicate injection to show reproducibility
0.0
0.2
0.4
0.6
0.8
1.0
1.0x104 1.0x105 1.0x106 1.0x107 1.0x108
Cum
ulat
ive
Wei
ght F
ract
ion
W
Molar Mass (g/mol)
Cumulative Molar Mass Weyer_20_1mgmL_b_03Weyer_20_1mgmL_b_01
34Copyright of Wyatt Technology Corporation
Summary of Results from SEC-MALS Analysis
Duplicate injection to show reproducibility
10.0
100.0
1000.0
10.0 12.0 14.0 16.0 18.0 20.0
R.M
.S. R
adiu
s (n
m)
Time (min)
RMS Radius vs. Time Weyer_20_1mgmL_b_03Weyer_20_1mgmL_b_01
35Copyright of Wyatt Technology Corporation
Summary of Results from SEC-MALS Analysis
Duplicate injection to show reproducibility
10.0
100.0
1000.0
1.0x104 1.0x105 1.0x106 1.0x107 1.0x108
R.M
.S. R
adiu
s (n
m)
Molar Mass (g/mol)
RMS Radius vs. Molar Mass Weyer_20_1mgmL_b_03Weyer_20_1mgmL_b_01
36Copyright of Wyatt Technology Corporation
Summary of Results from SEC-MALS Analysis
Chromatograms & Results forWeyco #21
37Copyright of Wyatt Technology Corporation
Summary of Results from SEC-MALS Analysis
Chromatograms: Weyco #21
-0.8
-0.7
-0.6
-0.5
-0.4
0 10 20 30
Det
ecto
r: A
UX1
Time (min)
0.24
0.28
0.32
0.36
0.40
0.44
Det
ecto
r: 1
1
Strip Chart - Weyer_21_1mgmL_b_01
38Copyright of Wyatt Technology Corporation
Summary of Results from SEC-MALS Analysis
Debye Plot of One Slice of the Above Chromatogram
90° & AUX detector
Peak, Slice : 1, 894 Time : 14.900 min Fit degree : 2 Conc. : (2.286 ± 0.004)e-5 g/mL Mw : (2.903 ± 0.055)e+5 g/mol Radius : 53.3 ± 4.0 nm
-31.70x10
-31.80x10
-31.90x10
-32.00x10
-32.10x10
-32.20x10
-32.30x10
0.0 0.2 0.4 0.6 0.8 1.0
sqrt[
K*c/
R(th
eta)
]
sin²(theta/2)
Debye Plot - Weyer_21_1mgmL_b_01
39Copyright of Wyatt Technology Corporation
Summary of Results from SEC-MALS Analysis
Distribution: MM vs. Elution Time
41.0x10
51.0x10
61.0x10
71.0x10
12.0 14.0 16.0 18.0
Mol
ar M
ass
(g/m
ol)
Time (min)
Molar Mass vs. Time Weyer_21_1mgmL_b_01
Mw = 2.2 x 105 (± 4%) PDI = 2.4 (± 9%)
40Copyright of Wyatt Technology Corporation
Summary of Results from SEC-MALS Analysis
Distribution: Cumulative MM Distribution
0.0
0.2
0.4
0.6
0.8
1.0
1.0x10 4 1.0x10 5 1.0x10 6 1.0x10 7
Cum
ulat
ive
Wei
ght F
ract
ion
W
Molar Mass (g/mol)
Cumulative Molar Mass Weyer_21_1mgmL_b_01
41Copyright of Wyatt Technology Corporation
Summary of Results from SEC-MALS Analysis
Distribution: RMS vs. Elution Time
10.0
100.0
1000.0
12.0 14.0 16.0 18.0
R.M
.S. R
adiu
s (n
m)
Time (min)
RMS Radius vs. Time Weyer_21_1mgmL_b_01
42Copyright of Wyatt Technology Corporation
Summary of Results from SEC-MALS Analysis
Conformational plot: Log(RMS) vs. Log(MM)
1.0
10.0
100.0
1000.0
1.0x10 4 1.0x10 5 1.0x10 6 1.0x10 7
R.M
.S. R
adiu
s (n
m)
Molar Mass (g/mol)
RMS Radius vs. Molar Mass Weyer_21_1mgmL_b_01
43Copyright of Wyatt Technology Corporation
Summary of Results from SEC-MALS Analysis
Duplicate injection to show reproducibility
41.0x10
51.0x10
61.0x10
71.0x10
81.0x10
12.0 14.0 16.0 18.0
Mol
ar M
ass
(g/m
ol)
Time (min)
Molar Mass vs. Time Weyer_21_1mgmL_b_01Weyer_21_1mgmL_b_02
44Copyright of Wyatt Technology Corporation
Summary of Results from SEC-MALS Analysis
Duplicate injection to show reproducibility
0.0
0.2
0.4
0.6
0.8
1.0
1.0x10 4 1.0x10 5 1.0x10 6 1.0x10 7 1.0x10 8
Cum
ulat
ive
Wei
ght F
ract
ion
W
Molar Mass (g/mol)
Cumulative Molar Mass Weyer_21_1mgmL_b_01Weyer_21_1mgmL_b_02
45Copyright of Wyatt Technology Corporation
Summary of Results from SEC-MALS Analysis
Duplicate injection to show reproducibility
10.0
100.0
1000.0
12.0 14.0 16.0 18.0
R.M
.S. R
adiu
s (n
m)
Time (min)
RMS Radius vs. Time Weyer_21_1mgmL_b_01Weyer_21_1mgmL_b_02
46Copyright of Wyatt Technology Corporation
Summary of Results from SEC-MALS Analysis
Duplicate injection to show reproducibility
1.0
10.0
100.0
1000.0
1.0x10 4 1.0x10 5 1.0x10 6 1.0x10 7
R.M
.S. R
adiu
s (n
m)
Molar Mass (g/mol)
RMS Radius vs. Molar Mass Weyer_21_1mgmL_b_01Weyer_21_1mgmL_b_02
47Copyright of Wyatt Technology Corporation
Summary of Results from SEC-MALS Analysis
RI Chromatograms Overlay
-0.1
0.0
0.1
0.2
0.3
0 10 20 30
LS, A
UX
(vol
ts)
Time (min)
Chromatograms Weyer_21_1mgmL_b_01Weyer_soft_1mgmL_b_01Weyer_9H4F_1mgmL_b_01Weyer_20_1mgmL_b_03
#20
#21
9H4F
Soft wood
48Copyright of Wyatt Technology Corporation
Summary of Results from SEC-MALS Analysis
LS Chromatograms Overlay
-0.1
0.0
0.1
0.2
0.3
0.4
0 10 20 30
LS, A
UX
(vol
ts)
Time (min)
Chromatograms Weyer_21_1mgmL_b_01Weyer_soft_1mgmL_b_01Weyer_9H4F_1mgmL_b_01Weyer_20_1mgmL_b_03
9H4F
Soft wood
#20
#21
49Copyright of Wyatt Technology Corporation
Summary of Results from SEC-MALS Analysis
Cumulative MM Distributions Overlay
0.0
0.2
0.4
0.6
0.8
1.0
1.0x10 4 1.0x10 5 1.0x10 6 1.0x10 7 1.0x10 8 1.0x10 9
Cum
ulat
ive
Wei
ght F
ract
ion
W
Molar Mass (g/mol)
Cumulative Molar Mass Weyer_21_1mgmL_b_01Weyer_soft_1mgmL_b_01Weyer_9H4F_1mgmL_b_01Weyer_20_1mgmL_b_03
9H4F
Soft wood
#21
#20
50Copyright of Wyatt Technology Corporation
Summary of Results from SEC-MALS Analysis
Mw results summarySample Mw (g/mol) PDI (Mw/Mn)
Softwood
1st injection
7.7 x 105 (± 10%) 2.9 (± 31%)
Softwood
2nd injection
7.9 x 105 (± 7%) 3.3 (± 18%)
9H4F
1st injection
3.1 x 106 (± 10%) 3.7 (± 14%)
9H4F
2nd injection
3.4 x 106 (± 15%) 3.8 (± 19%)
Weyco #20
1st injection
2.4 x 105 (± 3%) 3.0 (± 9%)
Weyco #20
2nd injection
2.0 x 105 (± 4%) 3.2 (± 9%)
Weyco #21
1st injection
2.2 x 105 (± 4%) 2.4 (± 9%)
Weyco #21
2nd injection
2.3 x105 (± 6%) 2.6 (± 13%)