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
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e-mail: [email protected]
1
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
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2
Summary of Results from SEC-MALS Analysis
Chromatograms & Results for
PA Softwood
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3
Summary of Results from SEC-MALS Analysis
Chromatograms: PA Softwood
Peak ID - Weyer_soft_1mgmL_b_01
0.4
LS #11
A UX 1
0.3
RI
L S # 1 1 , AUX1
LS
0.2
0.1
0.0
-0.1
0
10
20
Time (min)
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30
Summary of Results from SEC-MALS Analysis
Debye Plot of One Slice of the Above Chromatogram
Debye Plot - Weyer_soft_1mgmL_b_01
sqr t[K*c/R( theta) ]
2.40x 10
-3
2.00x 10 -3
1.60x 10 -3
1.20x 10 -3
8.00x 10 -4
0.0
0.2
0.4
0.6
0.8
s in²(theta/2)
Peak , Slic e : 1, 853
Time
: 14.217 min
Fit degree
:2
C onc . : (3.036 ± 0.006)e-5 g/mL
Mw
: (7.915 ± 0.644)e+5 g/mol
R adius : 97.0 ± 9.3 nm
90° & A UX detector
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1.0
Summary of Results from SEC-MALS Analysis
Distribution: MM vs. Elution Time
Molar Mas s v s . Time
Mo la r Ma ss ( g /mo l)
1.0x 10
W eyer_soft_1mgmL_b_01
7
Mw = 7.7 x 105 (± 10%)
PDI = 2.9 (± 31%)
1.0x 10 6
1.0x 10 5
1.0x 10 4
10.0
12.0
14.0
16.0
Time (min)
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18.0
20.0
Summary of Results from SEC-MALS Analysis
Distribution: Cumulative MM Distribution
C umulativ e Molar Mas s
W eyer_soft_1mgmL_b_01
1.0
Cu mu lative We ig h t Fr a ctio n W
0.8
0.6
0.4
0.2
0.0
1.0x 10
4
1.0x 10
5
1.0x 10
Molar Mas s (g/mol)
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6
1.0x 10
7
Summary of Results from SEC-MALS Analysis
Distribution: RMS vs. Elution Time
R MS R adius v s . Time
W eyer_soft_1mgmL_b_01
R.M.S. Ra d iu s ( n m)
1000.0
100.0
10.0
10.0
12.0
16.0
14.0
Time (min)
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18.0
20.0
Summary of Results from SEC-MALS Analysis
Conformational plot: Log(RMS) vs. Log(MM)
R MS R adius v s . Molar Mas s
W eyer_soft_1mgmL_b_01
R.M.S. Ra d iu s ( n m)
1000.0
100.0
Slope = 0.43
10.0
1.0x 10
4
1.0x 10
5
1.0x 10
Molar Mas s (g/mol)
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6
1.0x 10
7
Summary of Results from SEC-MALS Analysis
Duplicate injection to show reproducibility
Molar Mass vs. Time
Weyer_soft_1mgmL_b_01
Weyer_soft_1mgmL_b_02
Molar Mass (g/mol)
1.0x108
1.0x107
1.0x106
1.0x105
1.0x104
10.0
12.0
14.0
16.0
Time (min)
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10
18.0
20.0
Summary of Results from SEC-MALS Analysis
Duplicate injection to show reproducibility
Cumulative Molar Mass
Weyer_soft_1mgmL_b_01
Weyer_soft_1mgmL_b_02
Cumulative Weight Fraction W
1.0
0.8
0.6
0.4
0.2
0.0
1.0x10 4
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1.0x10 5
1.0x10 6
Molar Mass (g/mol)
11
1.0x10 7
1.0x10 8
Summary of Results from SEC-MALS Analysis
Duplicate injection to show reproducibility
RMS Radius vs. Time
Weyer_soft_1mgmL_b_01
Weyer_soft_1mgmL_b_02
R.M.S. Radius (nm)
1000.0
100.0
10.0
10.0
12.0
14.0
16.0
Time (min)
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18.0
20.0
Summary of Results from SEC-MALS Analysis
Duplicate injection to show reproducibility
RMS Radius vs. Molar Mass
Weyer_soft_1mgmL_b_01
Weyer_soft_1mgmL_b_02
R.M.S. Radius (nm)
1000.0
100.0
10.0
1.0x10 4
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1.0x10 5
1.0x10 6
Molar Mass (g/mol)
13
1.0x10 7
1.0x10 8
Summary of Results from SEC-MALS Analysis
Chromatograms & Results for
9H4F
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14
Summary of Results from SEC-MALS Analysis
Chromatograms: 9H4F
Strip C hart - W ey er_9H 4F_1mgmL_b_01
0.7
De te cto r : 1 1
0.6
0.5
0.4
0.3
De te cto r : AUX1
0.2
-0.8
-0.9
-1.0
-1.1
-1.2
0
10
20
Time (min)
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30
Summary of Results from SEC-MALS Analysis
Debye Plot of One Slice of the Above Chromatogram
Debye Plot - Weyer_9H4F_1mgmL_b_01
sq r t[K*c/R( the ta ) ]
1.80x 10
-3
1.60x 10 -3
1.40x 10 -3
1.20x 10 -3
1.00x 10 -3
8.00x 10 -4
0.0
0.2
0.4
0.6
0.8
s in²(theta/2)
Peak , Slic e : 1, 843
Time
: 14.050 min
Fit degree
:2
C onc . : (2.966 ± 0.005)e-5 g/mL
Mw
: (1.070 ± 0.012)e+6 g/mol
R adius : 113.5 ± 1.5 nm
90° & A UX detector
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1.0
Summary of Results from SEC-MALS Analysis
Distribution: MM vs. Elution Time
Molar Mas s v s . Time
1.0x 10
Mw = 3.1 x 106 (± 10%)
PDI = 3.7 (± 14%)
1.0x 10 8
Mo la r Ma ss ( g /mo l)
W eyer_9H4F_1mgmL_b_01
9
1.0x 10 7
1.0x 10 6
1.0x 10 5
10.0
12.0
14.0
Time (min)
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16.0
18.0
Summary of Results from SEC-MALS Analysis
Distribution: Cumulative MM Distribution
C umulativ e Molar Mas s
W eyer_9H4F_1mgmL_b_01
1.0
Cu mu lative We ig h t Fr a ctio n W
0.8
0.6
0.4
0.2
0.0
1.0x 10
5
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1.0x 10
6
1.0x 10 7
Molar Mas s (g/mol)
18
1.0x 10
8
1.0x 10
9
Summary of Results from SEC-MALS Analysis
Distribution: RMS vs. Elution Time
R MS R adius v s . Time
W eyer_9H4F_1mgmL_b_01
R.M.S. Ra d iu s ( n m)
10000.0
1000.0
100.0
10.0
10.0
12.0
14.0
Time (min)
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16.0
18.0
Summary of Results from SEC-MALS Analysis
Conformational plot: Log(RMS) vs. Log(MM)
R MS R adius v s . Molar Mas s
W eyer_9H4F_1mgmL_b_01
R.M.S. Ra d iu s ( n m)
10000.0
1000.0
100.0
Slope = 0.34
10.0
1.0x 10
5
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1.0x 10
6
1.0x 10 7
Molar Mas s (g/mol)
20
1.0x 10
8
1.0x 10
9
Summary of Results from SEC-MALS Analysis
Duplicate injection to show reproducibility
Molar Mas s v s . Time
1.0x 10
W eyer_9H4F_1mgmL_b_01
W eyer_9H4F_1mgmL_b_02
9
Mo la r Ma ss ( g /mo l)
1.0x 10 8
1.0x 10 7
1.0x 10 6
1.0x 10 5
10.0
12.0
14.0
Time (min)
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16.0
18.0
Summary of Results from SEC-MALS Analysis
Duplicate injection to show reproducibility
C umulativ e Molar Mas s
W eyer_9H4F_1mgmL_b_01
W eyer_9H4F_1mgmL_b_02
1.0
Cu mu lative We ig h t Fr a ctio n W
0.8
0.6
0.4
0.2
0.0
1.0x 10
5
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1.0x 10
6
1.0x 10 7
Molar Mas s (g/mol)
22
1.0x 10
8
1.0x 10
9
Summary of Results from SEC-MALS Analysis
Duplicate injection to show reproducibility
R MS R adius v s . Time
W eyer_9H4F_1mgmL_b_01
W eyer_9H4F_1mgmL_b_02
R.M.S. Ra d iu s ( n m)
10000.0
1000.0
100.0
10.0
10.0
12.0
14.0
Time (min)
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16.0
18.0
Summary of Results from SEC-MALS Analysis
Duplicate injection to show reproducibility
R MS R adius v s . Molar Mas s
W eyer_9H4F_1mgmL_b_01
W eyer_9H4F_1mgmL_b_02
R.M.S. Ra d iu s ( n m)
10000.0
1000.0
100.0
10.0
1.0x 10
5
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1.0x 10
6
1.0x 10 7
Molar Mas s (g/mol)
24
1.0x 10
8
1.0x 10
9
Summary of Results from SEC-MALS Analysis
Chromatograms & Results for
Weyco #20
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25
Summary of Results from SEC-MALS Analysis
Chromatograms: Weyco #20
Strip C hart - W ey er_20_1mgmL_b_03
De te cto r : 1 1
0.45
0.40
0.35
0.30
De te cto r : AUX1
0.25
-0.2
-0.3
-0.4
-0.5
-0.6
-0.7
0
10
20
Time (min)
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30
Summary of Results from SEC-MALS Analysis
Debye Plot of One Slice of the Above Chromatogram
Debye Plot - Weyer_20_1mgmL_b_03
sq r t[K*c/R( the ta ) ]
1.50x 10
-3
1.40x 10 -3
1.30x 10 -3
1.20x 10 -3
1.10x 10 -3
1.00x 10 -3
0.0
0.2
0.4
0.6
0.8
s in²(theta/2)
Peak , Slic e : 1, 853
Time
: 14.217 min
Fit degree
:2
C onc . : (1.146 ± 0.004)e-5 g/mL
Mw
: (8.617 ± 0.111)e+5 g/mol
R adius : 67.3 ± 2.4 nm
90° & A UX detector
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1.0
Summary of Results from SEC-MALS Analysis
Distribution: MM vs. Elution Time
Molar Mas s v s . Time
1.0x 10
Mw = 2.4 x 105 (± 3%)
PDI = 3.0 (± 9%)
1.0x 10 7
Mo la r Ma ss ( g /mo l)
W eyer_20_1mgmL_b_03
8
1.0x 10 6
1.0x 10 5
1.0x 10 4
10.0
12.0
14.0
16.0
Time (min)
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18.0
20.0
Summary of Results from SEC-MALS Analysis
Distribution: Cumulative MM Distribution
C umulativ e Molar Mas s
W eyer_20_1mgmL_b_03
1.0
Cu mu la tive We ig h t Fr a ctio n W
0.8
0.6
0.4
0.2
0.0
1.0x 10
4
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1.0x 10
5
1.0x 10 6
Molar Mas s (g/mol)
29
1.0x 10
7
1.0x 10
8
Summary of Results from SEC-MALS Analysis
Distribution: RMS vs. Elution Time
R MS R adius v s . Time
W eyer_20_1mgmL_b_03
R.M.S. Ra d iu s ( n m)
1000.0
100.0
10.0
10.0
12.0
16.0
14.0
Time (min)
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30
18.0
20.0
Summary of Results from SEC-MALS Analysis
Conformational plot: Log(RMS) vs. Log(MM)
R MS R adius v s . Molar Mas s
W eyer_20_1mgmL_b_03
R.M.S. Ra d iu s ( n m)
1000.0
100.0
10.0
1.0x 10
4
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1.0x 10
5
1.0x 10 6
Molar Mas s (g/mol)
31
1.0x 10
7
1.0x 10
8
Summary of Results from SEC-MALS Analysis
Duplicate injection to show reproducibility
Molar Mass vs. Time
Weyer_20_1mgmL_b_03
Weyer_20_1mgmL_b_01
Molar Mass (g/mol)
1.0x108
1.0x107
1.0x106
1.0x105
1.0x104
10.0
12.0
14.0
16.0
Time (min)
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32
18.0
20.0
Summary of Results from SEC-MALS Analysis
Duplicate injection to show reproducibility
Cumulative Molar Mass
Weyer_20_1mgmL_b_03
Weyer_20_1mgmL_b_01
Cumulative Weight Fraction W
1.0
0.8
0.6
0.4
0.2
0.0
1.0x10 4
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1.0x10 5
1.0x10 6
Molar Mass (g/mol)
33
1.0x10 7
1.0x10 8
Summary of Results from SEC-MALS Analysis
Duplicate injection to show reproducibility
RMS Radius vs. Time
Weyer_20_1mgmL_b_03
Weyer_20_1mgmL_b_01
R.M.S. Radius (nm)
1000.0
100.0
10.0
10.0
12.0
14.0
16.0
Time (min)
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34
18.0
20.0
Summary of Results from SEC-MALS Analysis
Duplicate injection to show reproducibility
RMS Radius vs. Molar Mass
Weyer_20_1mgmL_b_03
Weyer_20_1mgmL_b_01
R.M.S. Radius (nm)
1000.0
100.0
10.0
1.0x10 4
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1.0x10 5
1.0x10 6
Molar Mass (g/mol)
35
1.0x10 7
1.0x10 8
Summary of Results from SEC-MALS Analysis
Chromatograms & Results for
Weyco #21
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36
Summary of Results from SEC-MALS Analysis
Chromatograms: Weyco #21
Strip C hart - W ey er_21_1mgmL_b_01
0.44
De te cto r : 1 1
0.40
0.36
0.32
0.28
De te cto r : AUX1
0.24
-0.4
-0.5
-0.6
-0.7
-0.8
0
10
20
Time (min)
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37
30
Summary of Results from SEC-MALS Analysis
Debye Plot of One Slice of the Above Chromatogram
Debye Plot - Weyer_21_1mgmL_b_01
sq r t[K*c/R( the ta ) ]
2.30x 10
-3
2.20x 10 -3
2.10x 10 -3
2.00x 10 -3
1.90x 10 -3
1.80x 10 -3
1.70x 10 -3
0.0
0.2
0.4
0.6
0.8
s in²(theta/2)
Peak , Slic e : 1, 894
Time
: 14.900 min
Fit degree
:2
C onc . : (2.286 ± 0.004)e-5 g/mL
Mw
: (2.903 ± 0.055)e+5 g/mol
R adius : 53.3 ± 4.0 nm
90° & A UX detector
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1.0
Summary of Results from SEC-MALS Analysis
Distribution: MM vs. Elution Time
Molar Mas s v s . Time
Mo la r Ma ss ( g /mo l)
1.0x 10
W eyer_21_1mgmL_b_01
7
Mw = 2.2 x 105 (± 4%)
PDI = 2.4 (± 9%)
1.0x 10 6
1.0x 10 5
1.0x 10 4
12.0
14.0
16.0
Time (min)
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39
18.0
Summary of Results from SEC-MALS Analysis
Distribution: Cumulative MM Distribution
C umulativ e Molar Mas s
W eyer_21_1mgmL_b_01
1.0
Cu mu la tive We ig h t Fr a ctio n W
0.8
0.6
0.4
0.2
0.0
1.0x 10
4
1.0x 10
5
1.0x 10
Molar Mas s (g/mol)
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40
6
1.0x 10
7
Summary of Results from SEC-MALS Analysis
Distribution: RMS vs. Elution Time
R MS R adius v s . Time
W eyer_21_1mgmL_b_01
R.M.S. Ra d iu s ( n m)
1000.0
100.0
10.0
12.0
14.0
16.0
Time (min)
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41
18.0
Summary of Results from SEC-MALS Analysis
Conformational plot: Log(RMS) vs. Log(MM)
R MS R adius v s . Molar Mas s
W eyer_21_1mgmL_b_01
R.M.S. Ra d iu s ( n m)
1000.0
100.0
10.0
1.0
1.0x 10
4
1.0x 10
5
1.0x 10
Molar Mas s (g/mol)
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42
6
1.0x 10
7
Summary of Results from SEC-MALS Analysis
Duplicate injection to show reproducibility
Molar Mas s v s . Time
1.0x 10
W eyer_21_1mgmL_b_01
W eyer_21_1mgmL_b_02
8
Mo la r Ma ss ( g /mo l)
1.0x 10 7
1.0x 10 6
1.0x 10 5
1.0x 10 4
12.0
16.0
14.0
Time (min)
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43
18.0
Summary of Results from SEC-MALS Analysis
Duplicate injection to show reproducibility
C umulativ e Molar Mas s
W eyer_21_1mgmL_b_01
W eyer_21_1mgmL_b_02
1.0
Cu mu la tive We ig h t Fr a ctio n W
0.8
0.6
0.4
0.2
0.0
1.0x 10
4
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1.0x 10
5
1.0x 10 6
Molar Mas s (g/mol)
44
1.0x 10
7
1.0x 10
8
Summary of Results from SEC-MALS Analysis
Duplicate injection to show reproducibility
R MS R adius v s . Time
W eyer_21_1mgmL_b_01
W eyer_21_1mgmL_b_02
R.M.S. Ra d iu s ( n m)
1000.0
100.0
10.0
12.0
14.0
16.0
Time (min)
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45
18.0
Summary of Results from SEC-MALS Analysis
Duplicate injection to show reproducibility
R MS R adius v s . Molar Mas s
W eyer_21_1mgmL_b_01
W eyer_21_1mgmL_b_02
R.M.S. Ra d iu s ( n m)
1000.0
100.0
10.0
1.0
1.0x 10
4
1.0x 10
5
1.0x 10
Molar Mas s (g/mol)
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6
1.0x 10
7
Summary of Results from SEC-MALS Analysis
RI Chromatograms Overlay
C hromatograms
W eyer_21_1mgmL_b_01
W eyer_soft_1mgmL_b_01
W eyer_9H4F_1mgmL_b_01
W eyer_20_1mgmL_b_03
0.3
#20
0.2
L S, AUX ( vo lts)
#21
0.1
9H4F
Soft wood
0.0
-0.1
0
10
20
Time (min)
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30
Summary of Results from SEC-MALS Analysis
LS Chromatograms Overlay
C hromatograms
W eyer_21_1mgmL_b_01
W eyer_soft_1mgmL_b_01
W eyer_9H4F_1mgmL_b_01
W eyer_20_1mgmL_b_03
0.4
0.3
9H4F
L S, AUX ( vo lts)
#20
0.2
Soft wood
#21
0.1
0.0
-0.1
0
10
20
Time (min)
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30
Summary of Results from SEC-MALS Analysis
Cumulative MM Distributions Overlay
C umulativ e Molar Mas s
W eyer_21_1mgmL_b_01
W eyer_soft_1mgmL_b_01
W eyer_9H4F_1mgmL_b_01
W eyer_20_1mgmL_b_03
1.0
9H4F
Cu mu la tive We ig h t Fr a ctio n W
0.8
#20
0.6
Soft wood
0.4
#21
0.2
0.0
1.0x 10
4
1.0x 10
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5
1.0x 10 6
Molar Mas s (g/mol)
49
1.0x 10
7
1.0x 10
8
1.0x 10
9
Summary of Results from SEC-MALS Analysis
Mw results summary
Sample
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%)
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50