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ELECTROPHORESISSupporting Information
for Electrophoresis
DOI 10.1002/elps.200700634
Candace A. Luces, Sayo O. Fakayode, Mark Lowry and Isiah M. Warner
Protein separations using polyelectrolyte multilayer coatings with molecular
micelles in open tubular capillary electrochromatography
ª 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com
Protein Separations Using Polyelectrolyte Multilayer Coatings with Molecular
Micelles in Open Tubular Capillary Electrochromatography
Supporting Information
Candace A. Luces1, Sayo O. Fakayode2, Mark Lowry1, and Isiah M. Warner1*
1Department of Chemistry, Louisiana State University, Baton Rouge, LA 70803
2Department of Chemistry, Winston-Salem State University, Winston-Salem, NC 27110
*Corresponding author. Dr. Isiah M. Warner
Boyd Professor and Phillip W. West Professor of Analytical Chemistry
Howard Hughes Medical Institute Professor
Mailing Address: Louisiana State University
Department of Chemistry, Room 432 Choppin Hall
Baton Rouge, LA. 70803
Tel.: (225) 578-3945; Fax: (225) 578-3971
email: [email protected]
Table of Contents
Figure S1 Protein Separations in an Uncoated Capillary 3 Figure S2 Response Surface Plots of Design Variables 4 Table S1 Experimental parameters and Resolution values for 5 poly-L-glutamic acid- lysine as the cationic polymer in the PEM coating Table S2 Experimental parameters and Resolution values for 6 poly-L-lysine-serine as the cationic polymer in the PEM coating Table S3 Experimental parameters and Resolution values for 7 poly-L-lysine as the cationic polymer in the PEM coating Table S4 Experimental parameters and Resolution values for 8 poly-L-ornithine as the cationic polymer in the PEM coating
2
Figure S1 Protein Separations in an Uncoated Capillary
0 1 2 3 4 5 6 7 8 9 10Minutes
Bare Capillary Run 1Run 2Run 3
0 1 2 3 4 5 6 7 8 9 10Minutes
Bare Capillary
0 1 2 3 4 5 6 7 8 9 10Minutes
Bare Capillary
0 1 2 3 4 5 6 7 8 9 10Minutes
0 1 2 3 4 5 6 7 8 9 100 1 2 3 4 5 6 7 8 9 10Minutes
Bare Capillary Run 1Run 2Run 3
Run 1Run 2Run 3
Run 1Run 2Run 3
Conditions: Bare Capillary (reverse polarity); Analytes: 1. α-chymotrypsinogen A; 2. ribonuclease A; 3. lysozyme; 4. cytochrome c; Temperature: 15ºC; Voltage: 15kV. Buffer: 20mM phosphate, pH4
3
Figure S2 Response Surface Plots of Design Variables A: Influence of Temperature and Voltage on Protein Resolution: Conditions: 4A: Coating: 1.5, 2.5, 3.5 bilayers of 0.01% (w/v), 0.02% (w/v), 0.03% (w/v) poly-L-glutamic acid-lysine; Anionic Polymer: 0.5% (w/v) poly-L-SULA; Analytes: 1. α-chymotrypsinogen A; 2. ribonuclease A; 3. lysozyme; 4. cytochrome c; Temperature: 15ºC, 25ºC, 35ºC; Voltage: 15kV, 20kV, 30kV. All other conditions same as in Figure 2. B: Effect of the Number of Bilayers and Cationic Polymer Concentration on Protein Resolution: Conditions: Same as in Figure A
15 2025
3035
Temperature
35
25
20
30
15
Voltage
15 2025
30
35
25
20
30
15 Temperature
Voltage
35
15 2025
3035
Temperature
35
20
15
Voltage25
30
# bilayers1.52.0
2.5
3.53.0
0.01
0.02
0.03[polymer]
4.293 6.128 7.866 9.800 11.636 13.471
# bilayers1.5
2.02.5
3.53.0
0.01
0.02
0.03
2.688 3.286 3.884 4.482 5.086 5.678
[polymer]
# bilayers1.5
2.02.5
3.53.0
0.01
0.02
0.03
[polymer]
1.193 1.540 1.888 2.235 2.538 2.930
Rs1 Rs2 Rs3
1.897 4.445 6.992 9.540 12.088 14.6363.255 3.681 4.108 4.535 4.962 5.388
A
B
-0.205 0.580 1.366 2.152 2.937 3.773
Rs1 Rs2 Rs3
4
Table S1 Experimental parameters and Resolution values for poly-L-glutamic acid-lysine as the cationic polymer in the PEM coating No. Temp Volt Bilayers [Polymer] pH EOF MT1 MT2 MT3 MT4 Rs1 Rs2 Rs3
(˚C) (kV) (%w/v) 1 15 15 2.5 0.02 4 5.45 9.93 11.42 17.21 19.75 7.42 20.92 7.83 2 35 15 2.5 0.02 4 4.10 7.43 8.55 13.26 16.45 3.75 11.58 5.68 3 15 30 2.5 0.02 4 2.59 4.50 5.13 7.99 8.52 5.69 18.54 2.90 4 35 30 2.5 0.02 4 1.87 3.71 5.65 0.00 0.00 9.29 0.00 0.00 5 15 20 1.5 0.02 4 4.53 8.22 9.51 16.62 17.18 3.53 12.04 0.86 6 35 20 1.5 0.02 4 3.28 6.41 7.26 10.36 12.00 1.71 6.33 2.83 7 15 20 3.5 0.02 4 4.57 8.16 9.18 13.79 16.39 1.52 4.09 1.71 8 35 20 3.5 0.02 4 3.35 7.14 8.29 0.00 0.00 1.65 0.00 0.00 9 15 20 2.5 0.02 3 3.68 7.86 9.08 12.37 0.00 5.95 6.96 0.00
10 35 20 2.5 0.02 3 2.49 5.85 6.67 9.34 0.00 5.07 10.33 0.00 11 15 20 2.5 0.02 5 4.37 7.00 7.89 12.20 12.86 6.78 21.68 3.62 12 35 20 2.5 0.02 5 3.38 4.96 5.55 8.28 0.00 4.58 13.36 0.00 13 15 20 2.5 0.01 4 4.34 7.67 8.66 12.83 15.43 1.81 5.06 3.04 14 35 20 2.5 0.01 4 3.38 6.94 7.89 0.00 0.00 1.31 0.00 0.00 15 15 20 2.5 0.03 4 4.34 7.72 8.84 13.99 14.78 3.86 14.41 1.89 16 35 20 2.5 0.03 4 3.15 5.91 6.90 11.03 14.37 2.73 7.27 5.03 17 25 15 1.5 0.02 4 4.37 7.93 9.21 15.14 18.30 2.33 9.03 3.09 18 25 30 1.5 0.02 4 1.97 3.71 5.88 6.74 0.00 4.11 1.44 0.00 19 25 15 3.5 0.02 4 5.29 10.59 12.07 18.73 24.09 1.56 5.20 3.14 20 25 30 3.5 0.02 4 2.41 5.13 9.69 0.00 0.00 8.24 0.00 0.00 21 25 15 2.5 0.02 3 4.04 9.11 10.49 14.51 0.00 5.21 11.78 0.00 22 25 30 2.5 0.02 3 1.83 4.04 4.66 6.38 0.00 4.07 7.17 0.00 23 25 15 2.5 0.02 5 4.89 7.52 8.42 13.58 15.46 3.62 14.55 3.43 24 25 30 2.5 0.02 5 2.13 3.32 3.77 6.17 6.40 2.81 12.07 0.88 25 25 15 2.5 0.01 4 5.12 9.86 11.25 0.00 0.00 1.84 0.00 0.00 26 25 30 2.5 0.01 4 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 27 25 15 2.5 0.03 4 4.57 8.09 9.28 14.18 16.02 7.32 14.95 5.00 28 25 30 2.5 0.03 4 2.22 4.44 7.02 8.25 0.00 7.55 2.55 0.00 29 25 20 1.5 0.02 3 3.02 6.77 7.82 10.19 0.00 5.76 9.91 0.00 30 25 20 3.5 0.02 3 2.95 6.64 7.63 10.49 0.00 4.62 10.96 0.00 31 25 20 1.5 0.02 5 3.87 6.10 7.03 12.21 13.56 3.41 10.12 2.06 32 25 20 3.5 0.02 5 4.14 6.21 6.94 10.86 17.90 3.93 13.79 12.9233 25 20 1.5 0.01 4 3.44 5.96 6.86 11.10 12.04 6.59 12.43 3.55 34 25 20 3.5 0.01 4 4.07 8.32 9.54 0.00 0.00 1.40 0.00 0.00 35 25 20 1.5 0.03 4 2.96 4.75 5.28 7.13 7.30 4.65 13.35 1.16 36 25 20 3.5 0.03 4 3.64 6.48 7.33 11.02 12.77 2.00 7.21 2.33 37 25 20 2.5 0.01 3 3.02 6.64 7.66 10.49 0.00 5.52 12.47 0.00 38 25 20 2.5 0.01 5 4.00 6.44 7.43 13.26 15.73 3.07 8.88 2.75 39 25 20 2.5 0.03 3 3.11 6.84 7.88 10.87 0.00 7.03 13.83 0.00 40 25 20 2.5 0.03 5 3.93 6.18 7.03 12.67 15.08 3.17 5.30 1.30 41 25 20 2.5 0.02 4 3.33 6.13 6.87 10.11 11.32 5.56 11.87 3.59 42 25 20 2.5 0.02 4 3.30 5.82 6.67 10.13 11.48 3.99 9.32 3.94 43 25 20 2.5 0.02 4 3.41 5.85 6.70 10.13 11.68 3.84 11.16 4.13
5
Table S2 Experimental parameters and Resolution values for poly-L-lysine-serine as the cationic polymer in the PEM coating No. Temp Volt Bilayers [Polymer] pH EOF MT1 MT2 MT3 MT4 Rs1 Rs2 Rs3 (˚C) (kV) (%w/v)
1 15 15 2.5 0.02 4 4.59 7.58 8.49 11.34 12.49 5.21 11.34 5.112 35 15 2.5 0.02 4 5.62 12.24 14.68 27.58 0.00 2.21 7.21 0.003 15 30 2.5 0.02 4 2.05 3.09 3.40 4.44 4.60 3.83 10.87 1.424 35 30 2.5 0.02 4 3.03 3.86 6.55 0.00 0.00 9.84 0.00 0.005 15 20 1.5 0.02 4 3.08 4.41 4.75 5.97 6.15 3.52 10.27 1.586 35 20 1.5 0.02 4 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.007 15 20 3.5 0.02 4 5.15 8.62 8.96 9.43 10.46 0.83 0.72 3.028 35 20 3.5 0.02 4 3.70 12.64 15.27 0.00 0.00 1.36 0.00 0.009 15 20 2.5 0.02 3 3.97 9.44 11.05 16.42 0.00 7.43 15.74 0.00
10 35 20 2.5 0.02 3 2.15 5.08 5.69 7.17 0.00 3.15 6.21 0.0011 15 20 2.5 0.02 5 4.74 7.06 7.89 10.42 13.23 1.98 1.69 1.2712 35 20 2.5 0.02 5 4.11 5.71 6.04 13.60 0.00 0.59 7.04 0.0013 15 20 2.5 0.01 4 3.37 5.11 5.61 7.81 7.98 4.97 9.33 0.6114 35 20 2.5 0.01 4 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.0015 15 20 2.5 0.03 4 3.22 4.36 5.27 6.90 7.01 9.19 12.41 0.9316 35 20 2.5 0.03 4 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.0017 25 15 1.5 0.02 4 3.89 6.18 6.84 9.91 10.17 2.70 8.16 0.6218 25 30 1.5 0.02 4 1.83 2.77 3.08 4.43 0.00 2.26 7.82 0.0019 25 15 3.5 0.02 4 4.76 8.19 9.51 17.51 0.00 1.38 6.26 0.0020 25 30 3.5 0.02 4 2.23 4.65 0.00 0.00 0.00 0.00 0.00 0.0021 25 15 2.5 0.02 3 4.73 14.48 18.47 0.00 0.00 6.69 0.00 0.0022 25 30 2.5 0.02 3 1.96 4.93 5.94 9.54 0.00 4.10 11.26 0.0023 25 15 2.5 0.02 5 5.88 9.08 10.20 14.46 18.65 1.97 3.20 2.0724 25 30 2.5 0.02 5 2.43 3.64 5.36 8.29 0.00 3.06 1.60 0.0025 25 15 2.5 0.01 4 8.13 11.97 13.47 15.20 0.00 0.89 0.78 0.0026 25 30 2.5 0.01 4 1.86 3.40 4.87 0.00 0.00 6.00 0.00 0.0027 25 15 2.5 0.03 4 3.81 6.15 6.83 9.36 9.64 4.89 13.52 1.3828 25 30 2.5 0.03 4 1.83 2.79 3.08 4.15 0.00 3.80 13.70 0.0029 25 20 1.5 0.02 3 2.98 6.40 7.52 10.59 0.00 6.39 12.52 0.0030 25 20 3.5 0.02 3 2.93 6.50 7.48 10.36 0.00 5.55 11.46 0.0031 25 20 1.5 0.02 5 2.14 6.33 7.56 11.24 0.00 1.82 5.77 0.0032 25 20 3.5 0.02 5 2.89 3.83 4.10 5.15 6.21 1.98 7.13 6.7933 25 20 1.5 0.01 4 2.78 4.18 4.60 6.10 6.19 4.88 17.61 0.6834 25 20 3.5 0.01 4 3.04 5.66 6.60 0.00 0.00 1.56 0.00 0.0035 25 20 1.5 0.03 4 3.06 5.04 5.55 7.41 0.00 2.34 3.32 0.0036 25 20 3.5 0.03 4 3.35 6.00 6.51 10.15 0.00 0.60 3.66 0.0037 25 20 2.5 0.01 3 3.15 7.12 8.24 11.91 0.00 7.04 11.96 0.0038 25 20 2.5 0.01 5 6.33 11.10 13.10 0.00 0.00 0.65 0.00 0.0039 25 20 2.5 0.03 3 3.21 7.29 8.51 12.23 0.00 4.56 9.70 0.0040 25 20 2.5 0.03 5 5.25 9.07 10.48 12.79 0.00 0.77 0.93 0.0041 25 20 2.5 0.02 4 3.58 9.31 12.17 0.00 0.00 3.20 0.00 0.0042 25 20 2.5 0.02 4 3.84 8.72 10.53 0.00 0.00 2.21 0.00 0.0043 25 20 2.5 0.02 4 3.65 8.75 10.49 0.00 0.00 3.20 0.00 0.00
6
Table S3 Experimental parameters and Resolution values for poly-L-lysine as the cationic polymer in the PEM coating
No. Temp Volt Bilayers [Polymer] pH EOF MT1 MT2 MT3 MT4 Rs1 Rs2 Rs3 (˚C) (kV) (%w/v)
1 15 15 2.5 0.02 4 4.80 7.28 7.98 10.59 11.15 4.27 8.76 2.232 35 15 2.5 0.02 4 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.003 15 30 2.5 0.02 4 2.25 3.45 3.83 5.22 5.33 3.93 12.25 0.944 35 30 2.5 0.02 4 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.005 15 20 1.5 0.02 4 4.01 4.82 5.03 6.24 7.41 0.84 4.29 3.216 35 20 1.5 0.02 4 1.93 3.50 3.64 4.50 5.32 0.64 3.97 3.657 15 20 3.5 0.02 4 3.61 5.72 6.38 8.79 9.01 5.59 12.42 1.338 35 20 3.5 0.02 4 3.22 5.22 5.83 8.07 0.00 1.95 5.97 0.009 15 20 2.5 0.02 3 3.43 6.73 7.54 9.64 0.00 4.30 8.79 0.00
10 35 20 2.5 0.02 3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.0011 15 20 2.5 0.02 5 4.32 5.64 6.01 7.91 10.16 1.94 4.85 4.5412 35 20 2.5 0.02 5 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.0013 15 20 2.5 0.01 4 3.13 4.42 4.73 5.82 6.06 2.49 8.02 1.6514 35 20 2.5 0.01 4 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.0015 15 20 2.5 0.03 4 3.37 4.01 4.21 5.02 5.76 0.75 2.98 2.1816 35 20 2.5 0.03 4 2.32 2.77 2.92 3.48 4.08 0.94 3.37 2.8417 25 15 1.5 0.02 4 4.20 6.01 6.55 8.99 9.51 1.99 4.76 1.0118 25 30 1.5 0.02 4 1.95 2.84 3.07 3.99 4.25 1.07 3.22 1.3119 25 15 3.5 0.02 4 4.27 6.77 7.53 10.66 11.42 2.95 10.20 1.9320 25 30 3.5 0.02 4 2.12 2.62 2.80 3.50 4.29 1.23 5.20 3.2421 25 15 2.5 0.02 3 3.81 7.82 8.79 11.38 0.00 3.81 8.40 0.0022 25 30 2.5 0.02 3 1.74 3.48 3.93 5.06 0.00 3.97 7.07 0.0023 25 15 2.5 0.02 5 4.76 6.60 7.22 10.96 0.00 1.23 4.18 0.0024 25 30 2.5 0.02 5 1.74 2.59 2.87 4.14 0.00 2.87 10.00 0.0025 25 15 2.5 0.01 4 4.89 6.86 7.47 10.25 14.28 1.60 7.28 4.1626 25 30 2.5 0.01 4 1.68 2.43 2.63 3.36 3.49 2.63 8.19 1.2727 25 15 2.5 0.03 4 4.37 6.25 8.41 0.00 0.00 1.58 0.00 0.0028 25 30 2.5 0.03 4 2.23 3.63 4.93 6.34 0.00 1.49 1.58 0.0029 25 20 1.5 0.02 3 2.77 5.59 6.27 8.05 0.00 4.12 9.19 0.0030 25 20 3.5 0.02 3 2.69 4.84 5.31 6.43 0.00 2.39 4.47 0.0031 25 20 1.5 0.02 5 3.72 4.50 4.72 5.90 7.35 1.59 6.49 4.9032 25 20 3.5 0.02 5 3.18 5.50 6.56 0.00 0.00 1.90 0.00 0.0033 25 20 1.5 0.01 4 2.23 3.06 3.27 3.95 4.03 2.66 7.32 0.8734 25 20 3.5 0.01 4 3.19 5.13 5.68 0.00 0.00 1.11 0.00 0.0035 25 20 1.5 0.03 4 3.33 5.74 6.42 9.87 11.29 1.12 5.39 2.0936 25 20 3.5 0.03 4 3.42 6.08 6.94 11.02 14.71 1.21 5.13 3.2537 25 20 2.5 0.01 3 2.59 4.90 5.46 6.79 0.00 3.97 7.47 0.0038 25 20 2.5 0.01 5 3.37 4.06 4.25 5.41 6.60 0.69 2.73 1.1739 25 20 2.5 0.03 3 2.49 4.70 5.18 6.44 0.00 3.17 6.92 0.0040 25 20 2.5 0.03 5 2.98 3.72 3.89 4.83 5.92 0.50 2.05 1.2441 25 20 2.5 0.02 4 3.13 3.93 4.20 5.38 7.03 1.43 5.29 3.7842 25 20 2.5 0.02 4 3.30 4.58 4.79 6.05 8.55 0.54 4.26 3.1743 25 20 2.5 0.02 4 3.19 3.20 3.29 4.24 5.45 0.53 2.60 1.86
7
Table S4 Experimental parameters and Resolution values for poly-L-ornithine as the cationic polymer in the PEM coating No. Temp Volt Bilayers [Polymer] pH EOF MT1 MT2 MT3 MT4 Rs1 Rs2 Rs3
(˚C) (kV) (%w/v) 1 15 15 2.5 0.02 4 4.84 7.45 8.20 11.09 11.30 6.14 23.12 1.072 35 15 2.5 0.02 4 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.003 15 30 2.5 0.02 4 2.23 3.41 3.77 5.13 5.18 5.17 11.01 0.374 35 30 2.5 0.02 4 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.005 15 20 1.5 0.02 4 3.57 5.56 6.16 8.45 8.54 6.18 12.92 0.496 35 20 1.5 0.02 4 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.007 15 20 3.5 0.02 4 3.89 6.23 6.86 9.33 9.99 2.03 4.05 1.108 35 20 3.5 0.02 4 3.27 7.30 8.97 0.00 0.00 1.61 0.00 0.009 15 20 2.5 0.02 3 3.36 6.31 7.08 9.00 0.00 5.12 9.02 0.00
10 35 20 2.5 0.02 3 2.75 5.86 6.60 8.78 0.00 2.02 4.66 0.0011 15 20 2.5 0.02 5 5.09 7.91 8.93 14.07 0.00 3.37 12.21 0.0012 35 20 2.5 0.02 5 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.0013 15 20 2.5 0.01 4 3.53 5.44 5.99 8.08 8.24 5.19 12.69 0.9414 35 20 2.5 0.01 4 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.0015 15 20 2.5 0.03 4 3.62 5.80 6.46 8.92 9.18 7.28 14.40 1.4716 35 20 2.5 0.03 4 2.94 5.37 6.10 0.00 0.00 1.45 0.00 0.0017 25 15 1.5 0.02 4 3.99 6.34 7.05 9.67 9.93 5.84 16.17 1.4718 25 30 1.5 0.02 4 1.99 3.73 5.02 5.26 0.00 5.45 0.94 0.0019 25 15 3.5 0.02 4 4.36 7.32 8.28 11.93 12.16 4.53 10.60 0.5720 25 30 3.5 0.02 4 2.11 4.09 5.59 6.11 0.00 5.49 1.66 0.0021 25 15 2.5 0.02 3 3.04 7.81 8.88 11.57 0.00 5.60 11.83 0.0022 25 30 2.5 0.02 3 1.22 3.70 4.18 5.46 0.00 2.86 5.42 0.0023 25 15 2.5 0.02 5 6.22 10.02 11.37 0.00 0.00 1.49 0.00 0.0024 25 30 2.5 0.02 5 4.25 0.00 0.00 16.80 0.00 0.00 0.00 0.0025 25 15 2.5 0.01 4 4.00 6.47 7.22 10.14 10.22 6.06 15.08 0.3926 25 30 2.5 0.01 4 1.55 3.03 3.40 4.74 4.89 5.13 15.53 1.4127 25 15 2.5 0.03 4 4.07 6.73 7.53 10.15 11.01 5.75 15.42 4.4228 25 30 2.5 0.03 4 1.84 3.04 3.39 4.62 4.88 4.46 14.11 2.5029 25 20 1.5 0.02 3 2.81 5.50 6.23 8.09 0.00 7.97 13.55 0.0030 25 20 3.5 0.02 3 2.43 5.35 6.00 7.72 0.00 4.83 13.04 0.0031 25 20 1.5 0.02 5 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.0032 25 20 3.5 0.02 5 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.0033 25 20 1.5 0.01 4 2.95 4.70 5.36 7.56 7.82 6.70 17.02 1.6234 25 20 3.5 0.01 4 3.27 6.16 7.20 9.65 0.00 2.01 3.30 0.0035 25 20 1.5 0.03 4 3.26 5.31 5.77 8.05 8.40 0.88 3.73 0.7636 25 20 3.5 0.03 4 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.0037 25 20 2.5 0.01 3 2.50 5.35 5.85 7.43 0.00 2.89 11.64 0.0038 25 20 2.5 0.01 5 4.73 8.10 9.57 0.00 0.00 2.50 0.00 0.0039 25 20 2.5 0.03 3 4.02 5.40 6.04 7.73 0.00 5.38 12.36 0.0040 25 20 2.5 0.03 5 4.60 8.07 9.52 0.00 0.00 1.59 0.00 0.0041 25 20 2.5 0.02 4 3.42 5.92 6.65 9.22 10.03 1.38 4.71 1.4142 25 20 2.5 0.02 4 3.45 6.07 6.83 9.81 10.90 1.18 5.04 1.7243 25 20 2.5 0.02 4 3.59 6.73 7.52 11.09 13.01 1.12 4.41 2.17
8
