User:Benjamin Friedel/Notebook/CHEM 471/2015/09/09

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UV-Vis and Fluorescence Analysis of Lysozyme and Protease Sample Prep

Objective

There are two objectives for 09/02/2015. First, to calculate the molar absorptivity of lysozyme through UV-Vis and Fluorescence spectroscopy. Second, to prepare protease samples (using Chymotrypsin) for use in experiments at a later date.

Procedure and Data

1) First, Lysozyme samples were prepared for spectral analysis. A 10mL solution of 50.88uM lysozyme (MW=14307g/mol) in HPLC grade water was made by dissolving 7.28mg lysozyme in 10ml HPLC grade water.

    1. Solution was measured in a volumetric flask after lysozyme mass was measured on milligram balance in weigh boat.
    2. Solution stored in 15mL falcon tube.
  2. Further Lysozyme Solutions were made in concentrations of 0.965uM, 1.93uM, 3.86uM, 7.225uM and 15.445uM
    1. First, 10mL of a 30.89uM lysozyme solution in HPLC grade water was made by adding 4.42mg lysozyme in 10ml water in a 10ml volumetric flask.
    2. Then, serial dilutions were carried out using a 5ml pipette, dividing concentration of original solution by 1/2 each dilution by removing 5mL of the solution and adding it to 5mL of HPLC grade water.This was carried out 5 times to reach the above lysozyme concentrations, achieving 5mL of each solution except for the 0.965uM solution of which there were 10mL.
      1. ex.) For the 15.445uM solution, 5mL of the 30.89uM solution were pipetted into a 15mL falcon tube containing 5mL of HPLC grade water.
    3. Solution were stored at room temperature in 15mL falcon tubes and labeled with green tape.


2) UV-Vis Data was then collected for the 0.965uM, 1.93uM, 3.86uM, 7.225uM and 15.445uM lysozyme samples as well as an HPLC water blank from 200nm-800nm.

    1. First a water blank was measured
    2. Then the spectrum of each of the above noted samples was measured
    3. The spectra for each sample was corrected for solvent and baseline
      1. For blank correction, the blank spectra was subtracted from each sample spectra according to wavelength
      2. for baseline correction, the average absorbance was calculated between 700-800nm (lowest activity ranges as protein absorbs most highly at 280nm) for each sample spectra individually and then subtracted from that respective spectra.

Table 1: UV-Vis Raw and Adjusted Data from 200nm-800nm

' HPLC water blank 0.956uM 0.956uM-blank 0.956uM-blank-baseline 1.93uM 1.93uM-blank 1.93uM-blank-baseline 3.86uM 3.86uM-blank 3.86uM-blank-baseline 7.225uM 7.225uM-blank 7.225uM-blank-baseline 15.445uM 15.445uM-blank 15.445uM-blank-baseline
Wavelength(nm) Abs. Abs. Abs. Abs. Abs. Abs. Abs. Abs. Abs. Abs. Abs. Abs. Abs. Abs. Abs. Abs.
200 0.091 0.686 0.595 0.55923762 1.305 1.214 1.147 2.556 2.465 2.387 4.798 4.707 4.639 5 4.909 4.84
201 0.084 0.634 0.55 0.51423762 1.203 1.119 1.052 2.356 2.272 2.194 4.798 4.714 4.646 5 4.916 4.847
202 0.08 0.582 0.502 0.46623762 1.104 1.024 0.957 2.165 2.085 2.007 4.023 3.943 3.875 5 4.92 4.851
203 0.075 0.536 0.461 0.42523762 1.016 0.941 0.874 1.987 1.912 1.834 3.898 3.823 3.755 5 4.925 4.856
204 0.071 0.495 0.424 0.38823762 0.94 0.869 0.802 1.832 1.761 1.683 3.573 3.502 3.434 5 4.929 4.86
205 0.068 0.459 0.391 0.35523762 0.874 0.806 0.739 1.698 1.63 1.552 3.273 3.205 3.137 5 4.932 4.863
206 0.065 0.428 0.363 0.32723762 0.817 0.752 0.685 1.58 1.515 1.437 3.056 2.991 2.923 5 4.935 4.866
207 0.062 0.4 0.338 0.30223762 0.763 0.701 0.634 1.474 1.412 1.334 2.846 2.784 2.716 5 4.938 4.869
208 0.06 0.376 0.316 0.28023762 0.717 0.657 0.59 1.384 1.324 1.246 2.67 2.61 2.542 5 4.94 4.871
209 0.057 0.355 0.298 0.26223762 0.679 0.622 0.555 1.306 1.249 1.171 2.532 2.475 2.407 5 4.943 4.874
210 0.056 0.339 0.283 0.24723762 0.647 0.591 0.524 1.246 1.19 1.112 2.409 2.353 2.285 5 4.944 4.875
211 0.054 0.325 0.271 0.23523762 0.62 0.566 0.499 1.192 1.138 1.06 2.306 2.252 2.184 5 4.946 4.877
212 0.052 0.313 0.261 0.22523762 0.597 0.545 0.478 1.148 1.096 1.018 2.219 2.167 2.099 4.498 4.446 4.377
213 0.052 0.304 0.252 0.21623762 0.578 0.526 0.459 1.111 1.059 0.981 2.149 2.097 2.029 4.498 4.446 4.377
214 0.051 0.297 0.246 0.21023762 0.564 0.513 0.446 1.084 1.033 0.955 2.097 2.046 1.978 4.498 4.447 4.378
215 0.05 0.29 0.24 0.20423762 0.552 0.502 0.435 1.062 1.012 0.934 2.056 2.006 1.938 4.198 4.148 4.079
216 0.049 0.286 0.237 0.20123762 0.544 0.495 0.428 1.044 0.995 0.917 2.023 1.974 1.906 4.198 4.149 4.08
217 0.048 0.283 0.235 0.19923762 0.536 0.488 0.421 1.031 0.983 0.905 1.996 1.948 1.88 4.102 4.054 3.985
218 0.047 0.28 0.233 0.19723762 0.529 0.482 0.415 1.018 0.971 0.893 1.975 1.928 1.86 4.102 4.055 3.986
219 0.046 0.276 0.23 0.19423762 0.522 0.476 0.409 1.006 0.96 0.882 1.95 1.904 1.836 3.956 3.91 3.841
220 0.046 0.273 0.227 0.19123762 0.515 0.469 0.402 0.99 0.944 0.866 1.922 1.876 1.808 3.848 3.802 3.733
221 0.046 0.268 0.222 0.18623762 0.505 0.459 0.392 0.972 0.926 0.848 1.885 1.839 1.771 3.761 3.715 3.646
222 0.045 0.262 0.217 0.18123762 0.492 0.447 0.38 0.947 0.902 0.824 1.835 1.79 1.722 3.656 3.611 3.542
223 0.045 0.254 0.209 0.17323762 0.477 0.432 0.365 0.917 0.872 0.794 1.777 1.732 1.664 3.502 3.457 3.388
224 0.045 0.246 0.201 0.16523762 0.459 0.414 0.347 0.882 0.837 0.759 1.704 1.659 1.591 3.372 3.327 3.258
225 0.044 0.236 0.192 0.15623762 0.439 0.395 0.328 0.843 0.799 0.721 1.626 1.582 1.514 3.203 3.159 3.09
226 0.043 0.224 0.181 0.14523762 0.416 0.373 0.306 0.795 0.752 0.674 1.531 1.488 1.42 3.005 2.962 2.893
227 0.043 0.211 0.168 0.13223762 0.39 0.347 0.28 0.744 0.701 0.623 1.429 1.386 1.318 2.784 2.741 2.672
228 0.042 0.197 0.155 0.11923762 0.362 0.32 0.253 0.686 0.644 0.566 1.315 1.273 1.205 2.556 2.514 2.445
229 0.042 0.181 0.139 0.10323762 0.331 0.289 0.222 0.622 0.58 0.502 1.189 1.147 1.079 2.3 2.258 2.189
230 0.041 0.164 0.123 0.08723762 0.298 0.257 0.19 0.557 0.516 0.438 1.059 1.018 0.95 2.038 1.997 1.928
231 0.04 0.147 0.107 0.07123762 0.265 0.225 0.158 0.489 0.449 0.371 0.926 0.886 0.818 1.776 1.736 1.667
232 0.039 0.131 0.092 0.05623762 0.235 0.196 0.129 0.427 0.388 0.31 0.806 0.767 0.699 1.539 1.5 1.431
233 0.038 0.117 0.079 0.04323762 0.206 0.168 0.101 0.37 0.332 0.254 0.694 0.656 0.588 1.325 1.287 1.218
234 0.038 0.104 0.066 0.03023762 0.18 0.142 0.075 0.318 0.28 0.202 0.593 0.555 0.487 1.13 1.092 1.023
235 0.037 0.093 0.056 0.02023762 0.159 0.122 0.055 0.276 0.239 0.161 0.512 0.475 0.407 0.972 0.935 0.866
236 0.036 0.083 0.047 0.01123762 0.141 0.105 0.038 0.241 0.205 0.127 0.443 0.407 0.339 0.842 0.806 0.737
237 0.036 0.077 0.041 0.00523762 0.127 0.091 0.024 0.213 0.177 0.099 0.39 0.354 0.286 0.738 0.702 0.633
238 0.035 0.071 0.036 0.00023762 0.115 0.08 0.013 0.191 0.156 0.078 0.345 0.31 0.242 0.65 0.615 0.546
239 0.035 0.065 0.03 -0.00576238 0.105 0.07 0.003 0.171 0.136 0.058 0.306 0.271 0.203 0.574 0.539 0.47
240 0.035 0.061 0.026 -0.00976238 0.097 0.062 -0.005 0.155 0.12 0.042 0.275 0.24 0.172 0.513 0.478 0.409
241 0.034 0.058 0.024 -0.01176238 0.09 0.056 -0.011 0.141 0.107 0.029 0.249 0.215 0.147 0.461 0.427 0.358
242 0.034 0.055 0.021 -0.01476238 0.085 0.051 -0.016 0.13 0.096 0.018 0.227 0.193 0.125 0.416 0.382 0.313
243 0.033 0.053 0.02 -0.01576238 0.08 0.047 -0.02 0.12 0.087 0.009 0.209 0.176 0.108 0.381 0.348 0.279
244 0.033 0.05 0.017 -0.01876238 0.076 0.043 -0.024 0.113 0.08 0.002 0.194 0.161 0.093 0.351 0.318 0.249
245 0.033 0.049 0.016 -0.01976238 0.073 0.04 -0.027 0.107 0.074 -0.004 0.182 0.149 0.081 0.328 0.295 0.226
246 0.032 0.047 0.015 -0.02076238 0.07 0.038 -0.029 0.102 0.07 -0.008 0.172 0.14 0.072 0.31 0.278 0.209
247 0.032 0.046 0.014 -0.02176238 0.068 0.036 -0.031 0.097 0.065 -0.013 0.165 0.133 0.065 0.295 0.263 0.194
248 0.032 0.045 0.013 -0.02276238 0.067 0.035 -0.032 0.095 0.063 -0.015 0.159 0.127 0.059 0.285 0.253 0.184
249 0.032 0.045 0.013 -0.02276238 0.066 0.034 -0.033 0.093 0.061 -0.017 0.155 0.123 0.055 0.278 0.246 0.177
250 0.032 0.044 0.012 -0.02376238 0.065 0.033 -0.034 0.091 0.059 -0.019 0.153 0.121 0.053 0.274 0.242 0.173
251 0.031 0.044 0.013 -0.02276238 0.064 0.033 -0.034 0.091 0.06 -0.018 0.152 0.121 0.053 0.273 0.242 0.173
252 0.031 0.043 0.012 -0.02376238 0.064 0.033 -0.034 0.091 0.06 -0.018 0.153 0.122 0.054 0.274 0.243 0.174
253 0.031 0.043 0.012 -0.02376238 0.064 0.033 -0.034 0.091 0.06 -0.018 0.154 0.123 0.055 0.277 0.246 0.177
254 0.03 0.044 0.014 -0.02176238 0.065 0.035 -0.032 0.093 0.063 -0.015 0.157 0.127 0.059 0.282 0.252 0.183
255 0.03 0.044 0.014 -0.02176238 0.065 0.035 -0.032 0.095 0.065 -0.013 0.161 0.131 0.063 0.289 0.259 0.19
256 0.03 0.044 0.014 -0.02176238 0.066 0.036 -0.031 0.097 0.067 -0.011 0.165 0.135 0.067 0.298 0.268 0.199
257 0.03 0.045 0.015 -0.02076238 0.068 0.038 -0.029 0.1 0.07 -0.008 0.17 0.14 0.072 0.309 0.279 0.21
258 0.03 0.046 0.016 -0.01976238 0.068 0.038 -0.029 0.102 0.072 -0.006 0.176 0.146 0.078 0.32 0.29 0.221
259 0.03 0.046 0.016 -0.01976238 0.07 0.04 -0.027 0.105 0.075 -0.003 0.182 0.152 0.084 0.332 0.302 0.233
260 0.03 0.046 0.016 -0.01976238 0.071 0.041 -0.026 0.108 0.078 0 0.188 0.158 0.09 0.344 0.314 0.245
261 0.03 0.047 0.017 -0.01876238 0.072 0.042 -0.025 0.111 0.081 0.003 0.195 0.165 0.097 0.357 0.327 0.258
262 0.03 0.048 0.018 -0.01776238 0.074 0.044 -0.023 0.115 0.085 0.007 0.202 0.172 0.104 0.371 0.341 0.272
263 0.029 0.049 0.02 -0.01576238 0.076 0.047 -0.02 0.118 0.089 0.011 0.21 0.181 0.113 0.386 0.357 0.288
264 0.029 0.049 0.02 -0.01576238 0.077 0.048 -0.019 0.122 0.093 0.015 0.216 0.187 0.119 0.401 0.372 0.303
265 0.029 0.05 0.021 -0.01476238 0.079 0.05 -0.017 0.125 0.096 0.018 0.224 0.195 0.127 0.415 0.386 0.317
266 0.029 0.051 0.022 -0.01376238 0.081 0.052 -0.015 0.129 0.1 0.022 0.231 0.202 0.134 0.43 0.401 0.332
267 0.029 0.051 0.022 -0.01376238 0.082 0.053 -0.014 0.133 0.104 0.026 0.239 0.21 0.142 0.445 0.416 0.347
268 0.029 0.052 0.023 -0.01276238 0.084 0.055 -0.012 0.136 0.107 0.029 0.246 0.217 0.149 0.461 0.432 0.363
269 0.029 0.053 0.024 -0.01176238 0.086 0.057 -0.01 0.14 0.111 0.033 0.253 0.224 0.156 0.474 0.445 0.376
270 0.029 0.054 0.025 -0.01076238 0.088 0.059 -0.008 0.143 0.114 0.036 0.26 0.231 0.163 0.488 0.459 0.39
271 0.028 0.054 0.026 -0.00976238 0.089 0.061 -0.006 0.146 0.118 0.04 0.265 0.237 0.169 0.499 0.471 0.402
272 0.028 0.054 0.026 -0.00976238 0.089 0.061 -0.006 0.148 0.12 0.042 0.27 0.242 0.174 0.509 0.481 0.412
273 0.029 0.055 0.026 -0.00976238 0.09 0.061 -0.006 0.15 0.121 0.043 0.275 0.246 0.178 0.517 0.488 0.419
274 0.028 0.056 0.028 -0.00776238 0.091 0.063 -0.004 0.151 0.123 0.045 0.277 0.249 0.181 0.524 0.496 0.427
275 0.028 0.056 0.028 -0.00776238 0.091 0.063 -0.004 0.152 0.124 0.046 0.279 0.251 0.183 0.528 0.5 0.431
276 0.028 0.055 0.027 -0.00876238 0.091 0.063 -0.004 0.152 0.124 0.046 0.28 0.252 0.184 0.53 0.502 0.433
277 0.028 0.055 0.027 -0.00876238 0.091 0.063 -0.004 0.153 0.125 0.047 0.281 0.253 0.185 0.531 0.503 0.434
278 0.028 0.056 0.028 -0.00776238 0.092 0.064 -0.003 0.154 0.126 0.048 0.283 0.255 0.187 0.535 0.507 0.438
279 0.028 0.056 0.028 -0.00776238 0.093 0.065 -0.002 0.155 0.127 0.049 0.285 0.257 0.189 0.539 0.511 0.442
280 0.028 0.056 0.028 -0.00776238 0.093 0.065 -0.002 0.156 0.128 0.05 0.287 0.259 0.191 0.543 0.515 0.446
281 0.027 0.056 0.029 -0.00676238 0.093 0.066 -0.001 0.156 0.129 0.051 0.287 0.26 0.192 0.544 0.517 0.448
282 0.027 0.056 0.029 -0.00676238 0.092 0.065 -0.002 0.155 0.128 0.05 0.286 0.259 0.191 0.541 0.514 0.445
283 0.028 0.055 0.027 -0.00876238 0.091 0.063 -0.004 0.153 0.125 0.047 0.282 0.254 0.186 0.533 0.505 0.436
284 0.028 0.054 0.026 -0.00976238 0.089 0.061 -0.006 0.149 0.121 0.043 0.274 0.246 0.178 0.518 0.49 0.421
285 0.027 0.053 0.026 -0.00976238 0.086 0.059 -0.008 0.143 0.116 0.038 0.263 0.236 0.168 0.495 0.468 0.399
286 0.027 0.051 0.024 -0.01176238 0.083 0.056 -0.011 0.136 0.109 0.031 0.25 0.223 0.155 0.469 0.442 0.373
287 0.027 0.05 0.023 -0.01276238 0.08 0.053 -0.014 0.131 0.104 0.026 0.238 0.211 0.143 0.443 0.416 0.347
288 0.028 0.049 0.021 -0.01476238 0.079 0.051 -0.016 0.127 0.099 0.021 0.23 0.202 0.134 0.428 0.4 0.331
289 0.027 0.049 0.022 -0.01376238 0.079 0.052 -0.015 0.126 0.099 0.021 0.228 0.201 0.133 0.424 0.397 0.328
290 0.027 0.048 0.021 -0.01476238 0.078 0.051 -0.016 0.125 0.098 0.02 0.227 0.2 0.132 0.422 0.395 0.326
291 0.027 0.048 0.021 -0.01476238 0.077 0.05 -0.017 0.123 0.096 0.018 0.222 0.195 0.127 0.411 0.384 0.315
292 0.027 0.046 0.019 -0.01676238 0.073 0.046 -0.021 0.115 0.088 0.01 0.207 0.18 0.112 0.383 0.356 0.287
293 0.027 0.043 0.016 -0.01976238 0.067 0.04 -0.027 0.104 0.077 -0.001 0.184 0.157 0.089 0.337 0.31 0.241
294 0.027 0.04 0.013 -0.02276238 0.061 0.034 -0.033 0.092 0.065 -0.013 0.159 0.132 0.064 0.288 0.261 0.192
295 0.027 0.037 0.01 -0.02576238 0.056 0.029 -0.038 0.079 0.052 -0.026 0.135 0.108 0.04 0.241 0.214 0.145
296 0.027 0.034 0.007 -0.02876238 0.051 0.024 -0.043 0.069 0.042 -0.036 0.115 0.088 0.02 0.202 0.175 0.106
297 0.027 0.032 0.005 -0.03076238 0.047 0.02 -0.047 0.061 0.034 -0.044 0.098 0.071 0.003 0.169 0.142 0.073
298 0.027 0.031 0.004 -0.03176238 0.043 0.016 -0.051 0.054 0.027 -0.051 0.084 0.057 -0.011 0.142 0.115 0.046
299 0.026 0.029 0.003 -0.03276238 0.041 0.015 -0.052 0.048 0.022 -0.056 0.073 0.047 -0.021 0.118 0.092 0.023
300 0.026 0.028 0.002 -0.03376238 0.039 0.013 -0.054 0.044 0.018 -0.06 0.063 0.037 -0.031 0.099 0.073 0.004
301 0.027 0.028 0.001 -0.03476238 0.037 0.01 -0.057 0.04 0.013 -0.065 0.056 0.029 -0.039 0.084 0.057 -0.012
302 0.027 0.027 0 -0.03576238 0.035 0.008 -0.059 0.037 0.01 -0.068 0.049 0.022 -0.046 0.07 0.043 -0.026
303 0.027 0.026 -0.001 -0.03676238 0.034 0.007 -0.06 0.035 0.008 -0.07 0.045 0.018 -0.05 0.061 0.034 -0.035
304 0.027 0.026 -0.001 -0.03676238 0.033 0.006 -0.061 0.033 0.006 -0.072 0.041 0.014 -0.054 0.054 0.027 -0.042
305 0.027 0.026 -0.001 -0.03676238 0.033 0.006 -0.061 0.031 0.004 -0.074 0.038 0.011 -0.057 0.049 0.022 -0.047
306 0.026 0.025 -0.001 -0.03676238 0.031 0.005 -0.062 0.03 0.004 -0.074 0.036 0.01 -0.058 0.044 0.018 -0.051
307 0.027 0.025 -0.002 -0.03776238 0.031 0.004 -0.063 0.03 0.003 -0.075 0.034 0.007 -0.061 0.041 0.014 -0.055
308 0.027 0.025 -0.002 -0.03776238 0.031 0.004 -0.063 0.029 0.002 -0.076 0.033 0.006 -0.062 0.039 0.012 -0.057
309 0.027 0.024 -0.003 -0.03876238 0.03 0.003 -0.064 0.028 0.001 -0.077 0.032 0.005 -0.063 0.038 0.011 -0.058
310 0.027 0.024 -0.003 -0.03876238 0.031 0.004 -0.063 0.028 0.001 -0.077 0.032 0.005 -0.063 0.036 0.009 -0.06
311 0.027 0.024 -0.003 -0.03876238 0.03 0.003 -0.064 0.028 0.001 -0.077 0.032 0.005 -0.063 0.035 0.008 -0.061
312 0.027 0.024 -0.003 -0.03876238 0.03 0.003 -0.064 0.028 0.001 -0.077 0.031 0.004 -0.064 0.035 0.008 -0.061
313 0.027 0.024 -0.003 -0.03876238 0.03 0.003 -0.064 0.028 0.001 -0.077 0.031 0.004 -0.064 0.035 0.008 -0.061
314 0.027 0.024 -0.003 -0.03876238 0.03 0.003 -0.064 0.028 0.001 -0.077 0.03 0.003 -0.065 0.034 0.007 -0.062
315 0.027 0.024 -0.003 -0.03876238 0.03 0.003 -0.064 0.028 0.001 -0.077 0.03 0.003 -0.065 0.034 0.007 -0.062
316 0.027 0.025 -0.002 -0.03776238 0.03 0.003 -0.064 0.028 0.001 -0.077 0.03 0.003 -0.065 0.034 0.007 -0.062
317 0.027 0.024 -0.003 -0.03876238 0.03 0.003 -0.064 0.028 0.001 -0.077 0.03 0.003 -0.065 0.034 0.007 -0.062
318 0.028 0.024 -0.004 -0.03976238 0.03 0.002 -0.065 0.028 0 -0.078 0.031 0.003 -0.065 0.034 0.006 -0.063
319 0.027 0.025 -0.002 -0.03776238 0.03 0.003 -0.064 0.028 0.001 -0.077 0.03 0.003 -0.065 0.034 0.007 -0.062
320 0.026 0.024 -0.002 -0.03776238 0.029 0.003 -0.064 0.027 0.001 -0.077 0.029 0.003 -0.065 0.033 0.007 -0.062
321 0.026 0.023 -0.003 -0.03876238 0.029 0.003 -0.064 0.027 0.001 -0.077 0.029 0.003 -0.065 0.033 0.007 -0.062
322 0.026 0.024 -0.002 -0.03776238 0.029 0.003 -0.064 0.026 0 -0.078 0.029 0.003 -0.065 0.032 0.006 -0.063
323 0.026 0.024 -0.002 -0.03776238 0.029 0.003 -0.064 0.027 0.001 -0.077 0.03 0.004 -0.064 0.033 0.007 -0.062
324 0.027 0.024 -0.003 -0.03876238 0.03 0.003 -0.064 0.027 0 -0.078 0.029 0.002 -0.066 0.033 0.006 -0.063
325 0.027 0.024 -0.003 -0.03876238 0.029 0.002 -0.065 0.027 0 -0.078 0.029 0.002 -0.066 0.032 0.005 -0.064
326 0.026 0.024 -0.002 -0.03776238 0.029 0.003 -0.064 0.027 0.001 -0.077 0.029 0.003 -0.065 0.032 0.006 -0.063
327 0.027 0.024 -0.003 -0.03876238 0.03 0.003 -0.064 0.027 0 -0.078 0.029 0.002 -0.066 0.033 0.006 -0.063
328 0.027 0.024 -0.003 -0.03876238 0.029 0.002 -0.065 0.028 0.001 -0.077 0.029 0.002 -0.066 0.033 0.006 -0.063
329 0.026 0.024 -0.002 -0.03776238 0.029 0.003 -0.064 0.028 0.002 -0.076 0.029 0.003 -0.065 0.032 0.006 -0.063
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727 0.037 0.034 -0.003 -0.03876238 0.035 -0.002 -0.069 0.039 0.002 -0.076 0.036 -0.001 -0.069 0.036 -0.001 -0.07
728 0.038 0.034 -0.004 -0.03976238 0.035 -0.003 -0.07 0.04 0.002 -0.076 0.037 -0.001 -0.069 0.036 -0.002 -0.071
729 0.038 0.035 -0.003 -0.03876238 0.036 -0.002 -0.069 0.041 0.003 -0.075 0.037 -0.001 -0.069 0.037 -0.001 -0.07
730 0.038 0.035 -0.003 -0.03876238 0.036 -0.002 -0.069 0.041 0.003 -0.075 0.037 -0.001 -0.069 0.037 -0.001 -0.07
731 0.039 0.035 -0.004 -0.03976238 0.036 -0.003 -0.07 0.041 0.002 -0.076 0.038 -0.001 -0.069 0.037 -0.002 -0.071
732 0.039 0.036 -0.003 -0.03876238 0.037 -0.002 -0.069 0.042 0.003 -0.075 0.038 -0.001 -0.069 0.038 -0.001 -0.07
733 0.04 0.036 -0.004 -0.03976238 0.038 -0.002 -0.069 0.043 0.003 -0.075 0.039 -0.001 -0.069 0.039 -0.001 -0.07
734 0.04 0.037 -0.003 -0.03876238 0.038 -0.002 -0.069 0.043 0.003 -0.075 0.039 -0.001 -0.069 0.039 -0.001 -0.07
735 0.041 0.037 -0.004 -0.03976238 0.039 -0.002 -0.069 0.043 0.002 -0.076 0.039 -0.002 -0.07 0.039 -0.002 -0.071
736 0.041 0.038 -0.003 -0.03876238 0.039 -0.002 -0.069 0.044 0.003 -0.075 0.04 -0.001 -0.069 0.04 -0.001 -0.07
737 0.042 0.038 -0.004 -0.03976238 0.04 -0.002 -0.069 0.044 0.002 -0.076 0.04 -0.002 -0.07 0.04 -0.002 -0.071
738 0.041 0.038 -0.003 -0.03876238 0.04 -0.001 -0.068 0.044 0.003 -0.075 0.041 0 -0.068 0.041 0 -0.069
739 0.042 0.038 -0.004 -0.03976238 0.04 -0.002 -0.069 0.044 0.002 -0.076 0.041 -0.001 -0.069 0.041 -0.001 -0.07
740 0.042 0.038 -0.004 -0.03976238 0.04 -0.002 -0.069 0.045 0.003 -0.075 0.041 -0.001 -0.069 0.041 -0.001 -0.07
741 0.042 0.038 -0.004 -0.03976238 0.04 -0.002 -0.069 0.044 0.002 -0.076 0.041 -0.001 -0.069 0.041 -0.001 -0.07
742 0.042 0.038 -0.004 -0.03976238 0.04 -0.002 -0.069 0.045 0.003 -0.075 0.041 -0.001 -0.069 0.041 -0.001 -0.07
743 0.042 0.039 -0.003 -0.03876238 0.04 -0.002 -0.069 0.045 0.003 -0.075 0.041 -0.001 -0.069 0.041 -0.001 -0.07
744 0.042 0.038 -0.004 -0.03976238 0.04 -0.002 -0.069 0.045 0.003 -0.075 0.041 -0.001 -0.069 0.041 -0.001 -0.07
745 0.042 0.038 -0.004 -0.03976238 0.04 -0.002 -0.069 0.045 0.003 -0.075 0.041 -0.001 -0.069 0.041 -0.001 -0.07
746 0.042 0.039 -0.003 -0.03876238 0.04 -0.002 -0.069 0.044 0.002 -0.076 0.041 -0.001 -0.069 0.041 -0.001 -0.07
747 0.042 0.039 -0.003 -0.03876238 0.04 -0.002 -0.069 0.045 0.003 -0.075 0.041 -0.001 -0.069 0.041 -0.001 -0.07
748 0.042 0.039 -0.003 -0.03876238 0.04 -0.002 -0.069 0.045 0.003 -0.075 0.041 -0.001 -0.069 0.041 -0.001 -0.07
749 0.042 0.038 -0.004 -0.03976238 0.041 -0.001 -0.068 0.045 0.003 -0.075 0.041 -0.001 -0.069 0.041 -0.001 -0.07
750 0.043 0.039 -0.004 -0.03976238 0.04 -0.003 -0.07 0.045 0.002 -0.076 0.041 -0.002 -0.07 0.041 -0.002 -0.071
751 0.042 0.039 -0.003 -0.03876238 0.04 -0.002 -0.069 0.045 0.003 -0.075 0.041 -0.001 -0.069 0.041 -0.001 -0.07
752 0.042 0.039 -0.003 -0.03876238 0.04 -0.002 -0.069 0.044 0.002 -0.076 0.041 -0.001 -0.069 0.041 -0.001 -0.07
753 0.043 0.039 -0.004 -0.03976238 0.041 -0.002 -0.069 0.046 0.003 -0.075 0.042 -0.001 -0.069 0.041 -0.002 -0.071
754 0.042 0.039 -0.003 -0.03876238 0.04 -0.002 -0.069 0.045 0.003 -0.075 0.042 0 -0.068 0.041 -0.001 -0.07
755 0.042 0.039 -0.003 -0.03876238 0.04 -0.002 -0.069 0.046 0.004 -0.074 0.041 -0.001 -0.069 0.041 -0.001 -0.07
756 0.042 0.039 -0.003 -0.03876238 0.04 -0.002 -0.069 0.046 0.004 -0.074 0.041 -0.001 -0.069 0.041 -0.001 -0.07
757 0.042 0.039 -0.003 -0.03876238 0.04 -0.002 -0.069 0.045 0.003 -0.075 0.041 -0.001 -0.069 0.041 -0.001 -0.07
758 0.042 0.039 -0.003 -0.03876238 0.04 -0.002 -0.069 0.045 0.003 -0.075 0.042 0 -0.068 0.042 0 -0.069
759 0.042 0.039 -0.003 -0.03876238 0.041 -0.001 -0.068 0.046 0.004 -0.074 0.042 0 -0.068 0.041 -0.001 -0.07
760 0.043 0.039 -0.004 -0.03976238 0.04 -0.003 -0.07 0.046 0.003 -0.075 0.041 -0.002 -0.07 0.041 -0.002 -0.071
761 0.042 0.039 -0.003 -0.03876238 0.04 -0.002 -0.069 0.046 0.004 -0.074 0.041 -0.001 -0.069 0.041 -0.001 -0.07
762 0.043 0.039 -0.004 -0.03976238 0.04 -0.003 -0.07 0.045 0.002 -0.076 0.042 -0.001 -0.069 0.041 -0.002 -0.071
763 0.042 0.039 -0.003 -0.03876238 0.04 -0.002 -0.069 0.045 0.003 -0.075 0.041 -0.001 -0.069 0.041 -0.001 -0.07
764 0.042 0.039 -0.003 -0.03876238 0.04 -0.002 -0.069 0.046 0.004 -0.074 0.041 -0.001 -0.069 0.041 -0.001 -0.07
765 0.043 0.039 -0.004 -0.03976238 0.041 -0.002 -0.069 0.046 0.003 -0.075 0.042 -0.001 -0.069 0.041 -0.002 -0.071
766 0.042 0.039 -0.003 -0.03876238 0.04 -0.002 -0.069 0.046 0.004 -0.074 0.042 0 -0.068 0.041 -0.001 -0.07
767 0.042 0.039 -0.003 -0.03876238 0.04 -0.002 -0.069 0.045 0.003 -0.075 0.041 -0.001 -0.069 0.041 -0.001 -0.07
768 0.043 0.039 -0.004 -0.03976238 0.04 -0.003 -0.07 0.046 0.003 -0.075 0.042 -0.001 -0.069 0.041 -0.002 -0.071
769 0.043 0.039 -0.004 -0.03976238 0.04 -0.003 -0.07 0.047 0.004 -0.074 0.041 -0.002 -0.07 0.041 -0.002 -0.071
770 0.042 0.039 -0.003 -0.03876238 0.04 -0.002 -0.069 0.046 0.004 -0.074 0.041 -0.001 -0.069 0.041 -0.001 -0.07
771 0.043 0.038 -0.005 -0.04076238 0.04 -0.003 -0.07 0.046 0.003 -0.075 0.042 -0.001 -0.069 0.041 -0.002 -0.071
772 0.042 0.039 -0.003 -0.03876238 0.039 -0.003 -0.07 0.046 0.004 -0.074 0.041 -0.001 -0.069 0.04 -0.002 -0.071
773 0.042 0.039 -0.003 -0.03876238 0.039 -0.003 -0.07 0.046 0.004 -0.074 0.041 -0.001 -0.069 0.04 -0.002 -0.071
774 0.042 0.038 -0.004 -0.03976238 0.04 -0.002 -0.069 0.047 0.005 -0.073 0.041 -0.001 -0.069 0.04 -0.002 -0.071
775 0.042 0.038 -0.004 -0.03976238 0.039 -0.003 -0.07 0.046 0.004 -0.074 0.041 -0.001 -0.069 0.04 -0.002 -0.071
776 0.042 0.038 -0.004 -0.03976238 0.039 -0.003 -0.07 0.045 0.003 -0.075 0.041 -0.001 -0.069 0.04 -0.002 -0.071
777 0.043 0.039 -0.004 -0.03976238 0.04 -0.003 -0.07 0.047 0.004 -0.074 0.041 -0.002 -0.07 0.04 -0.003 -0.072
778 0.042 0.039 -0.003 -0.03876238 0.039 -0.003 -0.07 0.046 0.004 -0.074 0.041 -0.001 -0.069 0.04 -0.002 -0.071
779 0.042 0.038 -0.004 -0.03976238 0.039 -0.003 -0.07 0.046 0.004 -0.074 0.041 -0.001 -0.069 0.04 -0.002 -0.071
780 0.042 0.038 -0.004 -0.03976238 0.039 -0.003 -0.07 0.046 0.004 -0.074 0.04 -0.002 -0.07 0.04 -0.002 -0.071
781 0.042 0.038 -0.004 -0.03976238 0.039 -0.003 -0.07 0.045 0.003 -0.075 0.041 -0.001 -0.069 0.039 -0.003 -0.072
782 0.041 0.037 -0.004 -0.03976238 0.038 -0.003 -0.07 0.045 0.004 -0.074 0.04 -0.001 -0.069 0.04 -0.001 -0.07
783 0.042 0.038 -0.004 -0.03976238 0.038 -0.004 -0.071 0.045 0.003 -0.075 0.04 -0.002 -0.07 0.04 -0.002 -0.071
784 0.041 0.038 -0.003 -0.03876238 0.038 -0.003 -0.07 0.045 0.004 -0.074 0.04 -0.001 -0.069 0.039 -0.002 -0.071
785 0.041 0.038 -0.003 -0.03876238 0.038 -0.003 -0.07 0.046 0.005 -0.073 0.04 -0.001 -0.069 0.039 -0.002 -0.071
786 0.041 0.037 -0.004 -0.03976238 0.038 -0.003 -0.07 0.045 0.004 -0.074 0.039 -0.002 -0.07 0.039 -0.002 -0.071
787 0.041 0.037 -0.004 -0.03976238 0.038 -0.003 -0.07 0.046 0.005 -0.073 0.039 -0.002 -0.07 0.039 -0.002 -0.071
788 0.041 0.038 -0.003 -0.03876238 0.038 -0.003 -0.07 0.046 0.005 -0.073 0.04 -0.001 -0.069 0.04 -0.001 -0.07
789 0.041 0.038 -0.003 -0.03876238 0.038 -0.003 -0.07 0.045 0.004 -0.074 0.039 -0.002 -0.07 0.039 -0.002 -0.071
790 0.041 0.037 -0.004 -0.03976238 0.038 -0.003 -0.07 0.046 0.005 -0.073 0.039 -0.002 -0.07 0.038 -0.003 -0.072
791 0.041 0.037 -0.004 -0.03976238 0.038 -0.003 -0.07 0.046 0.005 -0.073 0.039 -0.002 -0.07 0.038 -0.003 -0.072
792 0.041 0.038 -0.003 -0.03876238 0.038 -0.003 -0.07 0.046 0.005 -0.073 0.039 -0.002 -0.07 0.039 -0.002 -0.071
793 0.04 0.037 -0.003 -0.03876238 0.037 -0.003 -0.07 0.045 0.005 -0.073 0.039 -0.001 -0.069 0.038 -0.002 -0.071
794 0.04 0.037 -0.003 -0.03876238 0.037 -0.003 -0.07 0.044 0.004 -0.074 0.039 -0.001 -0.069 0.038 -0.002 -0.071
795 0.039 0.036 -0.003 -0.03876238 0.037 -0.002 -0.069 0.044 0.005 -0.073 0.038 -0.001 -0.069 0.037 -0.002 -0.071
796 0.04 0.036 -0.004 -0.03976238 0.037 -0.003 -0.07 0.043 0.003 -0.075 0.039 -0.001 -0.069 0.038 -0.002 -0.071
797 0.04 0.036 -0.004 -0.03976238 0.037 -0.003 -0.07 0.045 0.005 -0.073 0.038 -0.002 -0.07 0.038 -0.002 -0.071
798 0.039 0.036 -0.003 -0.03876238 0.037 -0.002 -0.069 0.043 0.004 -0.074 0.038 -0.001 -0.069 0.038 -0.001 -0.07
799 0.04 0.036 -0.004 -0.03976238 0.037 -0.003 -0.07 0.045 0.005 -0.073 0.038 -0.002 -0.07 0.038 -0.002 -0.071
800 0.04 0.036 -0.004 -0.03976238 0.037 -0.003 -0.07 0.045 0.005 -0.073 0.038 -0.002 -0.07 0.038 -0.002 -0.071


Table 2: Absorbance of Each Lysozyme Concentration Adjusted for HPLC Water Blank and Baseline at 280nm

Concentration ( uM) Absorption
0.956 -0.007762376
1.93 -0.002
3.86 0.05
7.225 0.191
15.445 0.446

Figure 1: Lysozyme Absorption at 280nm as a Function of Concentration


Description of image

From this we can see that our molar absorptivity for lysozyme would be .0325Lumol-1cm-1 or 32.5mol-1cm-1 as A-Ebc, with E being the slope as b=1.

3) Fluorescence data was then measured for the 0.965uM, 1.93uM, 3.86uM, 7.225uM and 15.445uM lysozyme samples as well as an HPLC water blank.

    1. First a water blank was measured
    2. Then the spectrum of each of the above noted samples was measured
    3. The spectra for each sample was corrected for solvent blank
      1. For blank correction, the blank spectra was subtracted from each sample spectra according to wavelength

Table 3: Fluorescence Raw and Adjusted Data from 300nm-500nm Wavelengths

Wavelength (nm) HPLC Water blank 0.965uM 0.965-blank 1.93uM 1.93-blank 3.86uM 3.86-blank 7.225uM 7.225-blank 15.445uM 15.445-blank
Emission Intensity Emission Intensity Emission Intensity Emission Intensity Emission Intensity Emission Intensity Emission Intensity Emission Intensity Emission Intensity Emission Intensity Emission Intensity
300 0.241 1.714 1.473 4.044 3.803 7.223 6.982 10.01 9.769 14.053 13.812
300.5 0.231798 2.03325 1.801452 4.319538 4.08774 7.736019 7.504221 11.304191 11.072393 15.803222 15.571424
301 0.225261 2.338888 2.113627 4.614535 4.389274 8.285435 8.060174 12.590322 12.365061 17.547442 17.322181
301.5 0.223047 2.635071 2.412024 4.949361 4.726314 8.903301 8.680254 13.889913 13.666866 19.315293 19.092246
302 0.228552 2.924992 2.69644 5.323778 5.095226 9.592161 9.363609 15.225661 14.997109 21.129509 20.900957
302.5 0.244522 3.212784 2.968262 5.739772 5.49525 10.375192 10.13067 16.603248 16.358726 23.008914 22.764392
303 0.267249 3.498136 3.230887 6.213206 5.945957 11.250088 10.982839 18.029729 17.76248 24.981096 24.713847
303.5 0.29549 3.782348 3.486858 6.743837 6.448347 12.221671 11.926181 19.515792 19.220302 27.07876 26.78327
304 0.330513 4.080415 3.749902 7.320252 6.989739 13.279723 12.94921 21.111722 20.781209 29.280986 28.950473
304.5 0.372482 4.40778 4.035298 7.943491 7.571009 14.416857 14.044375 22.836812 22.46433 31.628813 31.256331
305 0.419023 4.760684 4.341661 8.607499 8.188476 15.626171 15.207148 24.688707 24.269684 34.156151 33.737128
305.5 0.471697 5.145484 4.673787 9.310102 8.838405 16.909374 16.437677 26.640423 26.168726 36.849717 36.37802
306 0.528381 5.5616 5.033219 10.049318 9.520937 18.272295 17.743914 28.692961 28.16458 39.692439 39.164058
306.5 0.588148 6.018216 5.430068 10.819654 10.231506 19.712849 19.124701 30.882519 30.294371 42.72168 42.133532
307 0.651476 6.509498 5.858022 11.627992 10.976516 21.225987 20.574511 33.172505 32.521029 45.961683 45.310207
307.5 0.715064 7.028985 6.313921 12.486288 11.771224 22.815255 22.100191 35.581102 34.866038 49.374521 48.659457
308 0.777629 7.568474 6.790845 13.40593 12.628301 24.481414 23.703785 38.077647 37.300018 52.984636 52.207007
308.5 0.841886 8.136667 7.294781 14.385542 13.543656 26.221722 25.379836 40.687618 39.845732 56.74809 55.906204
309 0.910814 8.733959 7.823145 15.436765 14.525951 28.039348 27.128534 43.432797 42.521983 60.633703 59.722889
309.5 0.979747 9.348815 8.369068 16.551743 15.571996 29.930526 28.950779 46.336476 45.356729 64.700986 63.721239
310 1.052985 9.993592 8.940607 17.723677 16.670692 31.897695 30.84471 49.355132 48.302147 68.941426 67.888441
310.5 1.128943 10.657105 9.528162 18.951345 17.822402 33.931165 32.802222 52.486879 51.357936 73.304546 72.175603
311 1.203638 11.353547 10.149909 20.218586 19.014948 36.037246 34.833608 55.762869 54.559231 77.841888 76.63825
311.5 1.275243 12.081265 10.806022 21.516561 20.241318 38.248507 36.973264 59.123397 57.848154 82.575548 81.300305
312 1.34132 12.833839 11.492519 22.839916 21.498596 40.548694 39.207374 62.639971 61.298651 87.489119 86.147799
312.5 1.40193 13.600307 12.198377 24.199256 22.797326 42.92569 41.52376 66.286749 64.884819 92.622357 91.220427
313 1.457897 14.378439 12.920542 25.59086 24.132963 45.378449 43.920552 70.040078 68.582181 97.957017 96.49912
313.5 1.509168 15.166029 13.656861 27.030541 25.521373 47.930261 46.421093 73.937197 72.428029 103.471745 101.962577
314 1.550367 15.956243 14.405876 28.514118 26.963751 50.578133 49.027766 77.983355 76.432988 109.193128 107.642761
314.5 1.588591 16.761309 15.172718 30.036566 28.447975 53.278026 51.689435 82.149906 80.561315 115.083112 113.494521
315 1.622839 17.561858 15.939019 31.604185 29.981346 56.004031 54.381192 86.473877 84.851038 121.074565 119.451726
315.5 1.65391 18.368003 16.714093 33.199677 31.545767 58.741792 57.087882 90.905967 89.252057 127.150642 125.496732
316 1.676645 19.189059 17.512414 34.80228 33.125635 61.546736 59.870091 95.37375 93.697105 133.292232 131.615587
316.5 1.6881 20.027096 18.338996 36.411613 34.723513 64.41671 62.72861 99.91326 98.22516 139.454796 137.766696
317 1.687316 20.87433 19.187014 38.033591 36.346275 67.332325 65.645009 104.517087 102.829771 145.680434 143.993118
317.5 1.677851 21.723087 20.045236 39.664651 37.9868 70.268426 68.590575 109.123967 107.446116 152.002493 150.324642
318 1.659588 22.578469 20.918881 41.302072 39.642484 73.241 71.581412 113.809483 112.149895 158.365797 156.706209
318.5 1.627299 23.447834 21.820535 42.957462 41.330163 76.282643 74.655344 118.490301 116.863002 164.873876 163.246577
319 1.588279 24.344866 22.756587 44.643633 43.055354 79.370905 77.782626 123.136889 121.54861 171.52424 169.935961
319.5 1.54012 25.262456 23.722336 46.395009 44.854889 82.476069 80.935949 127.886727 126.346607 178.311649 176.771529
320 1.487784 26.17204 24.684256 48.197345 46.709561 85.580486 84.092702 132.708429 131.220645 185.261878 183.774094
320.5 1.431515 27.061038 25.629523 50.031867 48.600352 88.739111 87.307596 137.537459 136.105944 192.141519 190.710004
321 1.370547 27.943139 26.572592 51.899806 50.529259 91.962865 90.592318 142.371398 141.000851 198.965641 197.595094
321.5 1.303949 28.825355 27.521406 53.791944 52.487995 95.240965 93.937016 147.229535 145.925586 205.77401 204.470061
322 1.234534 29.679596 28.445062 55.680727 54.446193 98.555997 97.321463 152.071789 150.837255 212.529652 211.295118
322.5 1.163718 30.50611 29.342392 57.551065 56.387347 101.807071 100.643353 156.967597 155.803879 219.002715 217.838997
323 1.088592 31.321452 30.23286 59.407955 58.319363 105.029333 103.940741 161.811408 160.722816 225.306517 224.217925
323.5 1.013374 32.143539 31.130165 61.250589 60.237215 108.216203 107.202829 166.503724 165.49035 231.494407 230.481033
324 0.935516 32.998722 32.063206 63.092338 62.156822 111.295935 110.360419 171.187025 170.251509 237.596126 236.66061
324.5 0.858543 33.878885 33.020342 64.932341 64.073798 114.32607 113.467527 175.905241 175.046698 243.747562 242.889019
325 0.786717 34.756982 33.970265 66.76445 65.977733 117.288911 116.502194 180.566628 179.779911 249.734639 248.947922
325.5 0.721899 35.649098 34.927199 68.604459 67.88256 120.226812 119.504913 185.109834 184.387935 255.653893 254.931994
326 0.662493 36.549759 35.887266 70.440135 69.777642 123.16998 122.507487 189.636505 188.974012 261.613026 260.950533
326.5 0.608481 37.438986 36.830505 72.235667 71.627186 126.187625 125.579144 194.113282 193.504801 267.782319 267.173838
327 0.561378 38.302427 37.741049 74.017799 73.456421 129.204503 128.643125 198.611 198.049622 274.018876 273.457498
327.5 0.517635 39.144049 38.626414 75.808014 75.290379 132.178958 131.661323 203.068188 202.550553 280.325295 279.80766
328 0.477328 39.964845 39.487517 77.56434 77.087012 135.168428 134.6911 207.48086 207.003532 286.816561 286.339233
328.5 0.440311 40.776506 40.336195 79.294141 78.85383 138.126234 137.685923 211.943094 211.502783 293.341866 292.901555
329 0.404451 41.594963 41.190512 81.013649 80.609198 141.124212 140.719761 216.595542 216.191091 299.951529 299.547078
329.5 0.371818 42.406246 42.034428 82.745713 82.373895 144.111031 143.739213 221.309561 220.937743 306.357598 305.98578
330 0.342575 43.23272 42.890145 84.498626 84.156051 147.109101 146.766526 226.001771 225.659196 312.608981 312.266406
330.5 0.319803 44.090254 43.770451 86.289314 85.969511 150.123907 149.804104 230.832136 230.512333 318.755678 318.435875
331 0.303472 44.960347 44.656875 88.068401 87.764929 153.23406 152.930588 235.721319 235.417847 324.806829 324.503357
331.5 0.293388 45.82383 45.530442 89.821319 89.527931 156.409063 156.115675 240.59455 240.301162 330.797695 330.504307
332 0.287203 46.69796 46.410757 91.662854 91.375651 159.600202 159.312999 245.414284 245.127081 336.841457 336.554254
332.5 0.284443 47.590948 47.306505 93.539375 93.254932 162.791756 162.507313 250.12461 249.840167 343.163714 342.879271
333 0.2861 48.474547 48.188447 95.43013 95.14403 166.006709 165.720609 254.822204 254.536104 349.589072 349.302972
333.5 0.289189 49.358627 49.069438 97.374829 97.08564 169.251131 168.961942 259.475905 259.186716 356.050526 355.761337
334 0.294262 50.238124 49.943862 99.360932 99.06667 172.453367 172.159105 264.062773 263.768511 362.402779 362.108517
334.5 0.299687 51.146806 50.847119 101.300915 101.001228 175.646262 175.346575 268.557047 268.25736 368.767402 368.467715
335 0.305888 52.111671 51.805783 103.22362 102.917732 178.825965 178.520077 273.210854 272.904966 375.109536 374.803648
335.5 0.312726 53.100454 52.787728 105.115814 104.803088 182.021157 181.708431 277.988825 277.676099 381.305389 380.992663
336 0.31961 54.077822 53.758212 106.865303 106.545693 185.197714 184.878104 282.788945 282.469335 387.333443 387.013833
336.5 0.325969 55.037293 54.711324 108.554984 108.229015 188.357622 188.031653 287.48792 287.161951 393.10257 392.776601
337 0.332133 56.01137 55.679237 110.201862 109.869729 191.453135 191.121002 291.977576 291.645443 398.906062 398.573929
337.5 0.339752 56.964508 56.624756 111.773394 111.433642 194.609056 194.269304 296.403765 296.064013 404.642546 404.302794
338 0.347187 57.869486 57.522299 113.370228 113.023041 197.705086 197.357899 300.590277 300.24309 410.224634 409.877447
338.5 0.354729 58.690616 58.335887 115.023242 114.668513 200.708693 200.353964 304.552556 304.197827 415.441628 415.086899
339 0.36184 59.443958 59.082118 116.547721 116.185881 203.65315 203.29131 308.181091 307.819251 420.62858 420.26674
339.5 0.368037 60.163504 59.795467 117.998814 117.630777 206.501435 206.133398 311.780847 311.41281 425.822416 425.454379
340 0.371995 60.876663 60.504668 119.458452 119.086457 209.392859 209.020864 315.513079 315.141084 431.044814 430.672819
340.5 0.372619 61.566413 61.193794 120.825722 120.453103 212.187387 211.814768 319.430157 319.057538 436.140747 435.768128
341 0.371161 62.216235 61.845074 122.107321 121.73616 214.874762 214.503601 323.502902 323.131741 440.959416 440.588255
341.5 0.367424 62.875154 62.50773 123.405788 123.038364 217.354468 216.987044 327.505169 327.137745 445.873114 445.50569
342 0.364801 63.550169 63.185368 124.643201 124.2784 219.769151 219.40435 331.51191 331.147109 450.680387 450.315586
342.5 0.362544 64.237528 63.874984 125.909961 125.547417 221.924271 221.561727 335.321738 334.959194 455.339873 454.977329
343 0.360998 64.906196 64.545198 127.292411 126.931413 223.877914 223.516916 338.93173 338.570732 459.526271 459.165273
343.5 0.363582 65.556329 65.192747 128.581088 128.217506 225.622618 225.259036 342.080522 341.71694 463.356394 462.992812
344 0.36985 66.167929 65.798079 129.739049 129.369199 227.117762 226.747912 344.891935 344.522085 466.899503 466.529653
344.5 0.375878 66.778819 66.402941 130.786469 130.410591 228.687038 228.31116 347.366814 346.990936 470.273955 469.898077
345 0.379619 67.369149 66.98953 131.727633 131.348014 230.301144 229.921525 349.778372 349.398753 473.429169 473.04955
345.5 0.381009 67.921596 67.540587 132.537467 132.156458 231.955967 231.574958 352.321561 351.940552 476.218581 475.837572
346 0.380781 68.435974 68.055193 133.301394 132.920613 233.612248 233.231467 354.71599 354.335209 479.139436 478.758655
346.5 0.379489 68.90145 68.521961 133.984232 133.604743 235.391374 235.011885 356.932624 356.553135 481.922963 481.543474
347 0.376442 69.351444 68.975002 134.599693 134.223251 237.089954 236.713512 358.935173 358.558731 484.714909 484.338467
347.5 0.371793 69.74764 69.375847 135.316938 134.945145 238.727553 238.35576 360.831438 360.459645 487.529915 487.158122
348 0.36852 70.119142 69.750622 135.985622 135.617102 240.084952 239.716432 362.398125 362.029605 490.253073 489.884553
348.5 0.368683 70.437333 70.06865 136.609629 136.240946 240.995634 240.626951 363.823477 363.454794 492.791884 492.423201
349 0.370428 70.719375 70.348947 137.147764 136.777336 241.700033 241.329605 365.036545 364.666117 495.176924 494.806496
349.5 0.371883 70.977578 70.605695 137.59751 137.225627 242.263049 241.891166 366.107423 365.73554 497.182066 496.810183
350 0.371002 71.256414 70.885412 137.976468 137.605466 242.744036 242.373034 367.323661 366.952659 498.774158 498.403156
350.5 0.371108 71.546095 71.174987 138.361586 137.990478 243.129993 242.758885 368.66744 368.296332 500.322668 499.95156
351 0.372381 71.807204 71.434823 138.760988 138.388607 243.547647 243.175266 370.05862 369.686239 501.523618 501.151237
351.5 0.372891 72.069842 71.696951 139.068831 138.69594 243.943684 243.570793 371.438373 371.065482 502.522668 502.149777
352 0.372046 72.261051 71.889005 139.424944 139.052898 244.531851 244.159805 372.602579 372.230533 503.634323 503.262277
352.5 0.371036 72.409496 72.03846 139.719329 139.348293 245.129163 244.758127 373.370207 372.999171 504.868428 504.497392
353 0.371942 72.530773 72.158831 139.988722 139.61678 245.536615 245.164673 373.868403 373.496461 505.766006 505.394064
353.5 0.372674 72.588649 72.215975 140.177425 139.804751 245.947373 245.574699 374.190401 373.817727 506.402384 506.02971
354 0.373601 72.594528 72.220927 140.238144 139.864543 246.32961 245.956009 374.302935 373.929334 506.66038 506.286779
354.5 0.372428 72.612724 72.240296 140.189735 139.817307 246.579962 246.207534 374.196043 373.823615 506.589061 506.216633
355 0.370898 72.653629 72.282731 140.08061 139.709712 246.661435 246.290537 374.042081 373.671183 506.208645 505.837747
355.5 0.369204 72.671974 72.30277 139.936786 139.567582 246.532481 246.163277 374.03231 373.663106 505.391271 505.022067
356 0.367906 72.707963 72.340057 139.670159 139.302253 246.149481 245.781575 374.239922 373.872016 504.233793 503.865887
356.5 0.367337 72.694224 72.326887 139.422947 139.05561 245.689515 245.322178 374.500411 374.133074 503.039327 502.67199
357 0.368067 72.651944 72.283877 139.14136 138.773293 245.178133 244.810066 374.597356 374.229289 502.177858 501.809791
357.5 0.370435 72.617637 72.247202 138.864367 138.493932 244.496761 244.126326 374.438652 374.068217 501.34336 500.972925
358 0.372765 72.529968 72.157203 138.616267 138.243502 243.856885 243.48412 374.288043 373.915278 500.742477 500.369712
358.5 0.374523 72.394248 72.019725 138.384012 138.009489 243.327259 242.952736 373.976753 373.60223 500.405178 500.030655
359 0.375919 72.213766 71.837847 138.103666 137.727747 242.951969 242.57605 373.303745 372.927826 500.270283 499.894364
359.5 0.375539 72.006829 71.63129 137.82744 137.451901 242.765472 242.389933 372.314333 371.938794 499.991812 499.616273
360 0.372407 71.743635 71.371228 137.517548 137.145141 242.649965 242.277558 371.120216 370.747809 499.235268 498.862861
360.5 0.367333 71.458239 71.090906 137.079302 136.711969 242.390576 242.023243 369.740284 369.372951 498.040691 497.673358
361 0.361116 71.136387 70.775271 136.561813 136.200697 241.965253 241.604137 368.168357 367.807241 496.457534 496.096418
361.5 0.354592 70.752255 70.397663 135.970655 135.616063 241.361921 241.007329 366.545328 366.190736 494.664697 494.310105
362 0.348977 70.365007 70.01603 135.240119 134.891142 240.438008 240.089031 364.854178 364.505201 492.590181 492.241204
362.5 0.344424 69.970161 69.625737 134.374969 134.030545 239.356345 239.011921 363.265015 362.920591 490.416984 490.07256
363 0.340666 69.609714 69.269048 133.457689 133.117023 238.090117 237.749451 361.655524 361.314858 488.4337 488.093034
363.5 0.338177 69.249191 68.911014 132.455915 132.117738 236.647176 236.308999 360.00418 359.666003 486.419915 486.081738
364 0.337989 68.887772 68.549783 131.481903 131.143914 235.197234 234.859245 358.417684 358.079695 484.321056 483.983067
364.5 0.338189 68.523873 68.185684 130.492829 130.15464 233.722744 233.384555 356.881066 356.542877 481.959303 481.621114
365 0.337824 68.168923 67.831099 129.44648 129.108656 232.162369 231.824545 355.173649 354.835825 479.425932 479.088108
365.5 0.336966 67.821551 67.484585 128.424359 128.087393 230.545175 230.208209 353.256018 352.919052 476.732871 476.395905
366 0.335171 67.417953 67.082782 127.458805 127.123634 228.887191 228.55202 351.165441 350.83027 473.759583 473.424412
366.5 0.330991 66.981353 66.650362 126.450223 126.119232 227.253824 226.922833 348.964178 348.633187 470.520962 470.189971
367 0.327906 66.511565 66.183659 125.405391 125.077485 225.734219 225.406313 346.649057 346.321151 467.146052 466.818146
367.5 0.323983 66.044464 65.720481 124.439364 124.115381 224.209755 223.885772 344.114227 343.790244 463.980857 463.656874
368 0.318844 65.531352 65.212508 123.498353 123.179509 222.606442 222.287598 341.28894 340.970096 460.75566 460.436816
368.5 0.313968 64.927241 64.613273 122.639059 122.325091 220.956033 220.642065 338.366612 338.052644 457.610265 457.296297
369 0.310341 64.291334 63.980993 121.778764 121.468423 219.249169 218.938828 335.417469 335.107128 454.297527 453.987186
369.5 0.307127 63.650484 63.343357 120.810948 120.503821 217.437411 217.130284 332.439232 332.132105 450.883458 450.576331
370 0.305673 63.018059 62.712386 119.795361 119.489688 215.520228 215.214555 329.255405 328.949732 447.338686 447.033013
370.5 0.305727 62.382573 62.076846 118.746984 118.441257 213.39891 213.093183 325.94662 325.640893 443.705327 443.3996
371 0.304421 61.755015 61.450594 117.540609 117.236188 211.145544 210.841123 322.695302 322.390881 439.893452 439.589031
371.5 0.304052 61.157028 60.852976 116.217675 115.913623 208.873845 208.569793 319.479842 319.17579 435.725337 435.421285
372 0.304116 60.599396 60.29528 114.833849 114.529733 206.628029 206.323913 316.374089 316.069973 431.5312 431.227084
372.5 0.304467 60.056198 59.751731 113.411744 113.107277 204.327994 204.023527 313.310297 313.00583 427.234798 426.930331
373 0.303769 59.462235 59.158466 112.087699 111.78393 202.002618 201.698849 310.35809 310.054321 423.002866 422.699097
373.5 0.30252 58.856185 58.553665 110.827144 110.524624 199.568918 199.266398 307.453498 307.150978 418.723526 418.421006
374 0.297634 58.255279 57.957645 109.581462 109.283828 197.102967 196.805333 304.600177 304.302543 414.447378 414.149744
374.5 0.291205 57.636931 57.345726 108.382178 108.090973 194.708998 194.417793 301.625682 301.334477 410.199402 409.908197
375 0.285347 57.00377 56.718423 107.252474 106.967127 192.304304 192.018957 298.475716 298.190369 406.078814 405.793467
375.5 0.278794 56.359559 56.080765 106.088506 105.809712 189.969037 189.690243 295.193339 294.914545 401.982251 401.703457
376 0.27218 55.720465 55.448285 104.946968 104.674788 187.673467 187.401287 291.696288 291.424108 397.660687 397.388507
376.5 0.265485 55.07171 54.806225 103.796226 103.530741 185.478384 185.212899 288.016449 287.750964 393.07731 392.811825
377 0.262183 54.41159 54.149407 102.585262 102.323079 183.380467 183.118284 284.249778 283.987595 388.351688 388.089505
377.5 0.260435 53.708887 53.448452 101.376171 101.115736 181.33444 181.074005 280.536841 280.276406 383.649589 383.389154
378 0.262169 52.983156 52.720987 100.133835 99.871666 179.145404 178.883235 277.020333 276.758164 378.977142 378.714973
378.5 0.264448 52.252562 51.988114 98.851584 98.587136 176.821647 176.557199 273.704128 273.43968 374.382293 374.117845
379 0.267181 51.504223 51.237042 97.478286 97.211105 174.484893 174.217712 270.406154 270.138973 369.724865 369.457684
379.5 0.269149 50.740045 50.470896 96.069811 95.800662 172.087723 171.818574 267.082602 266.813453 365.055355 364.786206
380 0.270462 49.978099 49.707637 94.578658 94.308196 169.687675 169.417213 263.759763 263.489301 360.399288 360.128826
380.5 0.270031 49.226474 48.956443 93.129213 92.859182 167.266442 166.996411 260.463435 260.193404 355.653277 355.383246
381 0.266458 48.48012 48.213662 91.729232 91.462774 164.833208 164.56675 257.041097 256.774639 350.784672 350.518214
381.5 0.263157 47.765471 47.502314 90.299282 90.036125 162.403081 162.139924 253.38959 253.126433 345.843386 345.580229
382 0.258768 47.072566 46.813798 88.884398 88.62563 160.04259 159.783822 249.732638 249.47387 340.975457 340.716689
382.5 0.255837 46.403884 46.148047 87.481811 87.225974 157.701096 157.445259 246.198457 245.94262 336.175521 335.919684
383 0.254182 45.750378 45.496196 86.108782 85.8546 155.351809 155.097627 242.677824 242.423642 331.563202 331.30902
383.5 0.252973 45.101318 44.848345 84.712538 84.459565 153.026722 152.773749 239.092339 238.839366 327.087722 326.834749
384 0.250253 44.431684 44.181431 83.31522 83.064967 150.715633 150.46538 235.442058 235.191805 322.539007 322.288754
384.5 0.248277 43.768957 43.52068 81.891341 81.643064 148.439666 148.191389 231.701246 231.452969 317.988866 317.740589
385 0.24691 43.09927 42.85236 80.478005 80.231095 146.188523 145.941613 227.96863 227.72172 313.363977 313.117067
385.5 0.244153 42.413973 42.16982 79.118049 78.873896 143.931574 143.687421 224.24786 224.003707 308.523632 308.279479
386 0.241519 41.740293 41.498774 77.757755 77.516236 141.650135 141.408616 220.394324 220.152805 303.476167 303.234648
386.5 0.23689 41.055341 40.818451 76.403205 76.166315 139.315775 139.078885 216.616045 216.379155 298.318515 298.081625
387 0.231405 40.366621 40.135216 75.076608 74.845203 136.973689 136.742284 212.99775 212.766345 293.005039 292.773634
387.5 0.226671 39.669535 39.442864 73.743325 73.516654 134.655454 134.428783 209.418753 209.192082 287.805393 287.578722
388 0.22335 38.967123 38.743773 72.395336 72.171986 132.352143 132.128793 205.863544 205.640194 282.782461 282.559111
388.5 0.219483 38.243141 38.023658 71.045884 70.826401 130.052117 129.832634 202.384722 202.165239 277.77936 277.559877
389 0.217998 37.529258 37.31126 69.695807 69.477809 127.737637 127.519639 198.816116 198.598118 273.108206 272.890208
389.5 0.218427 36.815574 36.597147 68.368203 68.149776 125.452514 125.234087 195.203036 194.984609 268.695623 268.477196
390 0.220571 36.105243 35.884672 67.093268 66.872697 123.189117 122.968546 191.575678 191.355107 264.398296 264.177725
390.5 0.222586 35.426485 35.203899 65.812875 65.590289 120.898743 120.676157 187.830616 187.60803 260.111126 259.88854
391 0.223659 34.762338 34.538679 64.543854 64.320195 118.527936 118.304277 184.244223 184.020564 255.881855 255.658196
391.5 0.224835 34.112049 33.887214 63.317009 63.092174 116.134316 115.909481 180.843646 180.618811 251.49278 251.267945
392 0.224717 33.474376 33.249659 62.09595 61.871233 113.761771 113.537054 177.433872 177.209155 246.894615 246.669898
392.5 0.222958 32.825863 32.602905 60.893196 60.670238 111.424517 111.201559 174.003033 173.780075 242.130613 241.907655
393 0.218389 32.154175 31.935786 59.669424 59.451035 109.124536 108.906147 170.669733 170.451344 237.153112 236.934723
393.5 0.212999 31.492301 31.279302 58.442356 58.229357 106.841398 106.628399 167.381846 167.168847 232.233238 232.020239
394 0.207047 30.846229 30.639182 57.222987 57.01594 104.599246 104.392199 164.052805 163.845758 227.433151 227.226104
394.5 0.203065 30.226801 30.023736 56.015538 55.812473 102.434746 102.231681 160.623064 160.419999 222.703512 222.500447
395 0.199762 29.649779 29.450017 54.817187 54.617425 100.369301 100.169539 157.103313 156.903551 218.040627 217.840865
395.5 0.197459 29.089115 28.891656 53.633864 53.436405 98.298842 98.101383 153.635306 153.437847 213.546854 213.349395
396 0.196459 28.532345 28.335886 52.498001 52.301542 96.235283 96.038824 150.33419 150.137731 209.205808 209.009349
396.5 0.196023 27.994742 27.798719 51.399292 51.203269 94.194325 93.998302 147.056785 146.860762 204.90805 204.712027
397 0.196498 27.449236 27.252738 50.357355 50.160857 92.209361 92.012863 143.83646 143.639962 200.580185 200.383687
397.5 0.197307 26.884843 26.687536 49.3463 49.148993 90.28342 90.086113 140.709034 140.511727 196.29561 196.098303
398 0.196956 26.312121 26.115165 48.358625 48.161669 88.37397 88.177014 137.74134 137.544384 192.127899 191.930943
398.5 0.195823 25.748297 25.552474 47.439516 47.243693 86.439994 86.244171 134.923101 134.727278 188.081331 187.885508
399 0.194707 25.188177 24.99347 46.549303 46.354596 84.534368 84.339661 132.149877 131.95517 184.200456 184.005749
399.5 0.192423 24.640907 24.448484 45.66522 45.472797 82.73863 82.546207 129.431356 129.238933 180.355962 180.163539
400 0.189316 24.096797 23.907481 44.771287 44.581971 80.970929 80.781613 126.773396 126.58408 176.560737 176.371421
400.5 0.186599 23.545813 23.359214 43.863217 43.676618 79.204778 79.018179 124.221422 124.034823 172.858115 172.671516
401 0.184316 23.020603 22.836287 42.955787 42.771471 77.45078 77.266464 121.672897 121.488581 169.104028 168.919712
401.5 0.182411 22.504951 22.32254 42.050291 41.86788 75.712045 75.529634 119.151797 118.969386 165.290148 165.107737
402 0.181768 22.012362 21.830594 41.138748 40.95698 74.020459 73.838691 116.612083 116.430315 161.460604 161.278836
402.5 0.180402 21.536381 21.355979 40.240854 40.060452 72.418893 72.238491 114.087767 113.907365 157.707917 157.527515
403 0.179345 21.094426 20.915081 39.403568 39.224223 70.813907 70.634562 111.583046 111.403701 154.058622 153.879277
403.5 0.177252 20.66626 20.489008 38.60157 38.424318 69.212147 69.034895 109.088928 108.911676 150.541162 150.36391
404 0.174663 20.250654 20.075991 37.840699 37.666036 67.688586 67.513923 106.655867 106.481204 147.220218 147.045555
404.5 0.171977 19.858701 19.686724 37.109062 36.937085 66.234614 66.062637 104.230159 104.058182 144.069668 143.897691
405 0.17113 19.466078 19.294948 36.376302 36.205172 64.825616 64.654486 101.865222 101.694092 141.089498 140.918368
405.5 0.17346 19.064962 18.891502 35.650744 35.477284 63.447415 63.273955 99.541425 99.367965 138.18965 138.01619
406 0.177682 18.650175 18.472493 34.923349 34.745667 62.073825 61.896143 97.318363 97.140681 135.327353 135.149671
406.5 0.183255 18.24515 18.061895 34.171119 33.987864 60.71199 60.528735 95.17013 94.986875 132.434043 132.250788
407 0.187496 17.847904 17.660408 33.401384 33.213888 59.435838 59.248342 93.129455 92.941959 129.570836 129.38334
407.5 0.191804 17.473492 17.281688 32.652787 32.460983 58.202646 58.010842 91.118543 90.926739 126.744589 126.552785
408 0.195016 17.107265 16.912249 31.934982 31.739966 56.994123 56.799107 89.151944 88.956928 123.947292 123.752276
408.5 0.195631 16.729286 16.533655 31.256532 31.060901 55.793332 55.597701 87.228437 87.032806 121.239778 121.044147
409 0.191518 16.360066 16.168548 30.626373 30.434855 54.615815 54.424297 85.334891 85.143373 118.593798 118.40228
409.5 0.185078 15.99784 15.812762 30.020604 29.835526 53.43239 53.247312 83.433362 83.248284 116.023039 115.837961
410 0.179706 15.628447 15.448741 29.438228 29.258522 52.258675 52.078969 81.535592 81.355886 113.535999 113.356293
410.5 0.175366 15.259245 15.083879 28.888567 28.713201 51.080549 50.905183 79.696892 79.521526 111.148232 110.972866
411 0.172105 14.902929 14.730824 28.349795 28.17769 49.896812 49.724707 77.937829 77.765724 108.770454 108.598349
411.5 0.168445 14.564308 14.395863 27.81232 27.643875 48.754308 48.585863 76.289728 76.121283 106.422331 106.253886
412 0.165334 14.255232 14.089898 27.271793 27.106459 47.669186 47.503852 74.708178 74.542844 104.118023 103.952689
412.5 0.164049 13.971103 13.807054 26.722184 26.558135 46.653661 46.489612 73.194188 73.030139 101.865033 101.700984
413 0.164538 13.695407 13.530869 26.183196 26.018658 45.681651 45.517113 71.748312 71.583774 99.701741 99.537203
413.5 0.165184 13.434946 13.269762 25.663104 25.49792 44.754542 44.589358 70.361803 70.196619 97.568765 97.403581
414 0.165336 13.185642 13.020306 25.161909 24.996573 43.858005 43.692669 68.981331 68.815995 95.481597 95.316261
414.5 0.165487 12.925281 12.759794 24.660132 24.494645 42.986793 42.821306 67.60136 67.435873 93.449223 93.283736
415 0.167828 12.664712 12.496884 24.180629 24.012801 42.127732 41.959904 66.22849 66.060662 91.50729 91.339462
415.5 0.172016 12.405018 12.233002 23.702129 23.530113 41.268732 41.096716 64.87867 64.706654 89.62073 89.448714
416 0.175422 12.140917 11.965495 23.213281 23.037859 40.418137 40.242715 63.56506 63.389638 87.789835 87.614413
416.5 0.17698 11.878674 11.701694 22.713632 22.536652 39.598778 39.421798 62.287078 62.110098 85.931737 85.754757
417 0.177415 11.624665 11.44725 22.206993 22.029578 38.819464 38.642049 61.039118 60.861703 84.061463 83.884048
417.5 0.178109 11.386554 11.208445 21.705441 21.527332 38.069377 37.891268 59.811265 59.633156 82.262399 82.08429
418 0.178402 11.157434 10.979032 21.229602 21.0512 37.340542 37.16214 58.606049 58.427647 80.492202 80.3138
418.5 0.177909 10.937803 10.759894 20.787258 20.609349 36.636956 36.459047 57.409007 57.231098 78.78226 78.604351
419 0.175863 10.71747 10.541607 20.363463 20.1876 35.946715 35.770852 56.229015 56.053152 77.137324 76.961461
419.5 0.174925 10.50465 10.329725 19.98681 19.811885 35.265869 35.090944 55.051142 54.876217 75.579723 75.404798
420 0.174812 10.301324 10.126512 19.641235 19.466423 34.580581 34.405769 53.872817 53.698005 74.108908 73.934096
420.5 0.174587 10.099629 9.925042 19.317321 19.142734 33.876414 33.701827 52.696074 52.521487 72.755096 72.580509
421 0.174148 9.903409 9.729261 18.997837 18.823689 33.184547 33.010399 51.549209 51.375061 71.411554 71.237406
421.5 0.173708 9.708992 9.535284 18.67501 18.501302 32.502227 32.328519 50.430512 50.256804 70.049901 69.876193
422 0.173154 9.526338 9.353184 18.331536 18.158382 31.832656 31.659502 49.371601 49.198447 68.725587 68.552433
422.5 0.172392 9.352436 9.180044 17.993481 17.821089 31.164844 30.992452 48.356484 48.184092 67.369206 67.196814
423 0.17182 9.18252 9.0107 17.648667 17.476847 30.541309 30.369489 47.415917 47.244097 66.005104 65.833284
423.5 0.170172 9.013173 8.843001 17.284303 17.114131 29.942965 29.772793 46.533804 46.363632 64.665762 64.49559
424 0.169549 8.843318 8.673769 16.927955 16.758406 29.361083 29.191534 45.702902 45.533353 63.345275 63.175726
424.5 0.17036 8.677997 8.507637 16.582517 16.412157 28.784729 28.614369 44.888227 44.717867 62.050667 61.880307
425 0.170847 8.513688 8.342841 16.268087 16.09724 28.209086 28.038239 44.060041 43.889194 60.830328 60.659481
425.5 0.170816 8.339209 8.168393 15.964781 15.793965 27.645122 27.474306 43.231479 43.060663 59.607369 59.436553
426 0.171668 8.154962 7.983294 15.685908 15.51424 27.067421 26.895753 42.381384 42.209716 58.353932 58.182264
426.5 0.172651 7.97092 7.798269 15.412178 15.239527 26.479839 26.307188 41.511123 41.338472 57.126302 56.953651
427 0.173323 7.789397 7.616074 15.151645 14.978322 25.870978 25.697655 40.602914 40.429591 55.884612 55.711289
427.5 0.174311 7.608142 7.433831 14.886372 14.712061 25.272218 25.097907 39.723169 39.548858 54.627824 54.453513
428 0.174887 7.432996 7.258109 14.605185 14.430298 24.687255 24.512368 38.85492 38.680033 53.401155 53.226268
428.5 0.175054 7.265122 7.090068 14.30267 14.127616 24.120456 23.945402 38.04728 37.872226 52.219371 52.044317
429 0.175656 7.112031 6.936375 13.985699 13.810043 23.586157 23.410501 37.299851 37.124195 51.098958 50.923302
429.5 0.17715 6.974077 6.796927 13.675852 13.498702 23.099039 22.921889 36.577697 36.400547 50.042858 49.865708
430 0.177351 6.842703 6.665352 13.360342 13.182991 22.656178 22.478827 35.890505 35.713154 49.010677 48.833326
430.5 0.177094 6.711117 6.534023 13.075755 12.898661 22.241117 22.064023 35.22381 35.046716 48.001706 47.824612
431 0.175629 6.580747 6.405118 12.817567 12.641938 21.84635 21.670721 34.559536 34.383907 47.016892 46.841263
431.5 0.173414 6.453718 6.280304 12.588411 12.414997 21.455313 21.281899 33.852156 33.678742 46.049806 45.876392
432 0.171822 6.318818 6.146996 12.377115 12.205293 21.061444 20.889622 33.148545 32.976723 45.091477 44.919655
432.5 0.169375 6.186656 6.017281 12.176981 12.007606 20.646844 20.477469 32.418703 32.249328 44.146473 43.977098
433 0.164802 6.049459 5.884657 11.981965 11.817163 20.193678 20.028876 31.663193 31.498391 43.201908 43.037106
433.5 0.161387 5.91141 5.750023 11.788373 11.626986 19.718482 19.557095 30.905302 30.743915 42.280136 42.118749
434 0.161389 5.776325 5.614936 11.589969 11.42858 19.247283 19.085894 30.137213 29.975824 41.401626 41.240237
434.5 0.162519 5.643817 5.481298 11.368645 11.206126 18.774048 18.611529 29.391067 29.228548 40.541084 40.378565
435 0.164148 5.513529 5.349381 11.143736 10.979588 18.314649 18.150501 28.657586 28.493438 39.685713 39.521565
435.5 0.165175 5.3889 5.223725 10.915283 10.750108 17.875959 17.710784 27.958942 27.793767 38.8345 38.669325
436 0.16527 5.276248 5.110978 10.684513 10.519243 17.45922 17.29395 27.273699 27.108429 37.994794 37.829524
436.5 0.166147 5.162618 4.996471 10.452425 10.286278 17.072779 16.906632 26.630305 26.464158 37.164407 36.99826
437 0.164594 5.056164 4.89157 10.220302 10.055708 16.705272 16.540678 26.032074 25.86748 36.351175 36.186581
437.5 0.15941 4.945837 4.786427 10.0013 9.84189 16.337873 16.178463 25.457377 25.297967 35.528382 35.368972
438 0.152608 4.833787 4.681179 9.807576 9.654968 15.972132 15.819524 24.900638 24.74803 34.712296 34.559688
438.5 0.146839 4.716925 4.570086 9.629898 9.483059 15.612556 15.465717 24.344158 24.197319 33.925934 33.779095
439 0.142506 4.595642 4.453136 9.453358 9.310852 15.252622 15.110116 23.826065 23.683559 33.144917 33.002411
439.5 0.139671 4.473986 4.334315 9.281508 9.141837 14.902666 14.762995 23.300272 23.160601 32.370478 32.230807
440 0.1381 4.351024 4.212924 9.110176 8.972076 14.55857 14.42047 22.781194 22.643094 31.602403 31.464303
440.5 0.137776 4.241174 4.103398 8.939498 8.801722 14.21308 14.075304 22.250418 22.112642 30.846251 30.708475
441 0.141383 4.140192 3.998809 8.76515 8.623767 13.872654 13.731271 21.722439 21.581056 30.093888 29.952505
441.5 0.144747 4.052866 3.908119 8.583542 8.438795 13.54555 13.400803 21.213479 21.068732 29.386829 29.242082
442 0.146561 3.971137 3.824576 8.397596 8.251035 13.234213 13.087652 20.706386 20.559825 28.701244 28.554683
442.5 0.147074 3.897082 3.750008 8.216496 8.069422 12.932719 12.785645 20.211069 20.063995 28.043524 27.89645
443 0.146444 3.827473 3.681029 8.042445 7.896001 12.640304 12.49386 19.711102 19.564658 27.4097 27.263256
443.5 0.144614 3.757734 3.61312 7.872784 7.72817 12.349784 12.20517 19.253562 19.108948 26.800544 26.65593
444 0.143231 3.690782 3.547551 7.703542 7.560311 12.067866 11.924635 18.809233 18.666002 26.211553 26.068322
444.5 0.142458 3.618186 3.475728 7.536042 7.393584 11.803482 11.661024 18.377561 18.235103 25.639666 25.497208
445 0.141322 3.542795 3.401473 7.371767 7.230445 11.546465 11.405143 17.963831 17.822509 25.063644 24.922322
445.5 0.142596 3.467263 3.324667 7.206978 7.064382 11.289391 11.146795 17.571151 17.428555 24.459655 24.317059
446 0.142871 3.396635 3.253764 7.052337 6.909466 11.034301 10.89143 17.202805 17.059934 23.875094 23.732223
446.5 0.14376 3.321843 3.178083 6.902722 6.758962 10.786674 10.642914 16.838203 16.694443 23.306554 23.162794
447 0.144535 3.244584 3.100049 6.760773 6.616238 10.552477 10.407942 16.47959 16.335055 22.775281 22.630746
447.5 0.14405 3.169252 3.025202 6.628384 6.484334 10.327175 10.183125 16.124527 15.980477 22.261473 22.117423
448 0.140058 3.097379 2.957321 6.507249 6.367191 10.113661 9.973603 15.786481 15.646423 21.774905 21.634847
448.5 0.134928 3.030638 2.89571 6.390064 6.255136 9.902634 9.767706 15.452978 15.31805 21.317341 21.182413
449 0.130107 2.963436 2.833329 6.272898 6.142791 9.69941 9.569303 15.107347 14.97724 20.899345 20.769238
449.5 0.12569 2.895199 2.769509 6.153528 6.027838 9.500765 9.375075 14.769273 14.643583 20.496651 20.370961
450 0.12362 2.829324 2.705704 6.027215 5.903595 9.299178 9.175558 14.448781 14.325161 20.080969 19.957349
450.5 0.12165 2.766576 2.644926 5.902107 5.780457 9.093951 8.972301 14.143862 14.022212 19.663784 19.542134
451 0.121358 2.702926 2.581568 5.776199 5.654841 8.879343 8.757985 13.839795 13.718437 19.23597 19.114612
451.5 0.12371 2.637002 2.513292 5.651252 5.527542 8.662433 8.538723 13.536698 13.412988 18.805089 18.681379
452 0.127726 2.572945 2.445219 5.528463 5.400737 8.44857 8.320844 13.24166 13.113934 18.373074 18.245348
452.5 0.12918 2.511183 2.382003 5.413388 5.284208 8.244975 8.115795 12.946885 12.817705 17.956958 17.827778
453 0.128621 2.455453 2.326832 5.30644 5.177819 8.044594 7.915973 12.653587 12.524966 17.548163 17.419542
453.5 0.127061 2.404509 2.277448 5.21304 5.085979 7.858088 7.731027 12.356118 12.229057 17.17049 17.043429
454 0.124396 2.353308 2.228912 5.119316 4.99492 7.68864 7.564244 12.066343 11.941947 16.807409 16.683013
454.5 0.120768 2.302635 2.181867 5.025524 4.904756 7.538431 7.417663 11.796886 11.676118 16.457915 16.337147
455 0.115057 2.256183 2.141126 4.932731 4.817674 7.403724 7.288667 11.538076 11.423019 16.127911 16.012854
455.5 0.109699 2.212005 2.102306 4.8396 4.729901 7.272688 7.162989 11.294062 11.184363 15.798616 15.688917
456 0.106269 2.16559 2.059321 4.747361 4.641092 7.147958 7.041689 11.069739 10.96347 15.461571 15.355302
456.5 0.104884 2.119696 2.014812 4.656631 4.551747 7.021148 6.916264 10.863505 10.758621 15.119931 15.015047
457 0.104608 2.07295 1.968342 4.564393 4.459785 6.892557 6.787949 10.65541 10.550802 14.792868 14.68826
457.5 0.105958 2.025725 1.919767 4.471174 4.365216 6.750106 6.644148 10.443017 10.337059 14.469215 14.363257
458 0.10823 1.979651 1.871421 4.39261 4.28438 6.598314 6.490084 10.225838 10.117608 14.156946 14.048716
458.5 0.111584 1.932622 1.821038 4.315147 4.203563 6.440129 6.328545 10.012332 9.900748 13.849794 13.73821
459 0.112509 1.88825 1.775741 4.238628 4.126119 6.287152 6.174643 9.803061 9.690552 13.548789 13.43628
459.5 0.110564 1.848319 1.737755 4.165503 4.054939 6.139908 6.029344 9.594397 9.483833 13.266492 13.155928
460 0.107337 1.812232 1.704895 4.095596 3.988259 5.998609 5.891272 9.382381 9.275044 12.98965 12.882313
460.5 0.103632 1.78022 1.676588 4.030624 3.926992 5.871743 5.768111 9.185847 9.082215 12.718943 12.615311
461 0.098064 1.753873 1.655809 3.973367 3.875303 5.762399 5.664335 9.005024 8.90696 12.448622 12.350558
461.5 0.091398 1.729105 1.637707 3.916768 3.82537 5.664781 5.573383 8.826698 8.7353 12.189978 12.09858
462 0.086238 1.703874 1.617636 3.852803 3.766565 5.566949 5.480711 8.649068 8.56283 11.951732 11.865494
462.5 0.083283 1.679229 1.595946 3.792756 3.709473 5.471155 5.387872 8.474225 8.390942 11.72749 11.644207
463 0.084834 1.650765 1.565931 3.725643 3.640809 5.369943 5.285109 8.305142 8.220308 11.511942 11.427108
463.5 0.087382 1.620276 1.532894 3.652324 3.564942 5.266337 5.178955 8.137822 8.05044 11.302113 11.214731
464 0.089726 1.587388 1.497662 3.574561 3.484835 5.159191 5.069465 7.969741 7.880015 11.099427 11.009701
464.5 0.092708 1.55084 1.458132 3.49778 3.405072 5.049099 4.956391 7.800986 7.708278 10.897618 10.80491
465 0.095336 1.512421 1.417085 3.425784 3.330448 4.94138 4.846044 7.642719 7.547383 10.692595 10.597259
465.5 0.095976 1.473782 1.377806 3.365278 3.269302 4.839634 4.743658 7.490625 7.394649 10.483267 10.387291
466 0.09399 1.439567 1.345577 3.316317 3.222327 4.745137 4.651147 7.346521 7.252531 10.264016 10.170026
466.5 0.090498 1.411064 1.320566 3.273919 3.183421 4.647761 4.557263 7.220312 7.129814 10.057934 9.967436
467 0.086796 1.388671 1.301875 3.232453 3.145657 4.557805 4.471009 7.103315 7.016519 9.859264 9.772468
467.5 0.084668 1.368226 1.283558 3.18903 3.104362 4.475139 4.390471 6.996225 6.911557 9.675906 9.591238
468 0.08365 1.353595 1.269945 3.147103 3.063453 4.396376 4.312726 6.890356 6.806706 9.513829 9.430179
468.5 0.083877 1.341457 1.25758 3.100533 3.016656 4.314938 4.231061 6.785614 6.701737 9.369235 9.285358
469 0.085215 1.32746 1.242245 3.055874 2.970659 4.238876 4.153661 6.680655 6.59544 9.244495 9.15928
469.5 0.086624 1.310635 1.224011 3.012067 2.925443 4.16876 4.082136 6.581571 6.494947 9.12398 9.037356
470 0.087442 1.286096 1.198654 2.972592 2.88515 4.102112 4.01467 6.477173 6.389731 9.005117 8.917675
470.5 0.087902 1.261538 1.173636 2.939849 2.851947 4.044315 3.956413 6.369455 6.281553 8.867348 8.779446
471 0.086836 1.237685 1.150849 2.911629 2.824793 3.982841 3.896005 6.265521 6.178685 8.72609 8.639254
471.5 0.084699 1.212456 1.127757 2.876779 2.79208 3.922584 3.837885 6.163749 6.07905 8.576797 8.492098
472 0.082035 1.187069 1.105034 2.831494 2.749459 3.867193 3.785158 6.063035 5.981 8.424534 8.342499
472.5 0.079202 1.165741 1.086539 2.787914 2.708712 3.812161 3.732959 5.961652 5.88245 8.282925 8.203723
473 0.077538 1.149507 1.071969 2.740362 2.662824 3.750914 3.673376 5.856902 5.779364 8.14367 8.066132
473.5 0.076996 1.135963 1.058967 2.691465 2.614469 3.689712 3.612716 5.753216 5.67622 8.014836 7.93784
474 0.077467 1.124832 1.047365 2.643794 2.566327 3.626629 3.549162 5.658951 5.581484 7.89325 7.815783
474.5 0.077394 1.109002 1.031608 2.601289 2.523895 3.562375 3.484981 5.566747 5.489353 7.781103 7.703709
475 0.078557 1.094735 1.016178 2.571581 2.493024 3.50329 3.424733 5.474199 5.395642 7.667822 7.589265
475.5 0.081072 1.081535 1.000463 2.55192 2.470848 3.444979 3.363907 5.382296 5.301224 7.550396 7.469324
476 0.082832 1.065496 0.982664 2.531475 2.448643 3.388122 3.30529 5.301017 5.218185 7.422898 7.340066
476.5 0.083098 1.04697 0.963872 2.504803 2.421705 3.335241 3.252143 5.227389 5.144291 7.302013 7.218915
477 0.082523 1.027164 0.944641 2.480674 2.398151 3.291482 3.208959 5.155138 5.072615 7.198735 7.116212
477.5 0.082591 1.007323 0.924732 2.45545 2.372859 3.252002 3.169411 5.085266 5.002675 7.099115 7.016524
478 0.081909 0.989143 0.907234 2.42968 2.347771 3.215858 3.133949 5.018255 4.936346 7.001949 6.92004
478.5 0.0809 0.973354 0.892454 2.404356 2.323456 3.176445 3.095545 4.958068 4.877168 6.916981 6.836081
479 0.07908 0.957897 0.878817 2.379455 2.300375 3.135794 3.056714 4.907095 4.828015 6.839887 6.760807
479.5 0.076659 0.945676 0.869017 2.364848 2.288189 3.093705 3.017046 4.858003 4.781344 6.767306 6.690647
480 0.076272 0.935497 0.859225 2.355429 2.279157 3.05035 2.974078 4.803622 4.72735 6.694711 6.618439
480.5 0.074426 0.926002 0.851576 2.345201 2.270775 3.007163 2.932737 4.757615 4.683189 6.612657 6.538231
481 0.072125 0.918301 0.846176 2.330621 2.258496 2.963062 2.890937 4.713423 4.641298 6.530074 6.457949
481.5 0.069559 0.911399 0.84184 2.312179 2.24262 2.924292 2.854733 4.664414 4.594855 6.450526 6.380967
482 0.067773 0.904471 0.836698 2.289224 2.221451 2.895079 2.827306 4.60685 4.539077 6.373684 6.305911
482.5 0.065922 0.89664 0.830718 2.263807 2.197885 2.872859 2.806937 4.543268 4.477346 6.299136 6.233214
483 0.064724 0.88667 0.821946 2.234906 2.170182 2.850302 2.785578 4.473668 4.408944 6.229722 6.164998
483.5 0.064889 0.876728 0.811839 2.200258 2.135369 2.829106 2.764217 4.408878 4.343989 6.158192 6.093303
484 0.063948 0.867439 0.803491 2.169798 2.10585 2.805534 2.741586 4.344413 4.280465 6.086715 6.022767
484.5 0.064583 0.85485 0.790267 2.145287 2.080704 2.779804 2.715221 4.277265 4.212682 6.014202 5.949619
485 0.064009 0.84084 0.776831 2.121537 2.057528 2.752895 2.688886 4.218881 4.154872 5.937835 5.873826
485.5 0.064643 0.825958 0.761315 2.103259 2.038616 2.721192 2.656549 4.168304 4.103661 5.873851 5.809208
486 0.066108 0.811122 0.745014 2.086251 2.020143 2.687226 2.621118 4.128233 4.062125 5.813181 5.747073
486.5 0.068161 0.798821 0.73066 2.070989 2.002828 2.65634 2.588179 4.092333 4.024172 5.757264 5.689103
487 0.070126 0.786967 0.716841 2.058376 1.98825 2.629232 2.559106 4.061081 3.990955 5.71116 5.641034
487.5 0.071301 0.775375 0.704074 2.045972 1.974671 2.604352 2.533051 4.032446 3.961145 5.671963 5.600662
488 0.072878 0.766198 0.69332 2.029892 1.957014 2.581274 2.508396 4.008126 3.935248 5.63279 5.559912
488.5 0.072788 0.760072 0.687284 2.012953 1.940165 2.555965 2.483177 3.98083 3.908042 5.59072 5.517932
489 0.072334 0.755532 0.683198 1.999528 1.927194 2.531326 2.458992 3.954601 3.882267 5.535734 5.4634
489.5 0.068607 0.751542 0.682935 1.981887 1.91328 2.510179 2.441572 3.923733 3.855126 5.468444 5.399837
490 0.066046 0.747299 0.681253 1.96461 1.898564 2.489841 2.423795 3.884455 3.818409 5.405712 5.339666
490.5 0.065106 0.742297 0.677191 1.943104 1.877998 2.469419 2.404313 3.843859 3.778753 5.340091 5.274985
491 0.065232 0.74002 0.674788 1.924001 1.858769 2.44936 2.384128 3.796521 3.731289 5.271697 5.206465
491.5 0.065791 0.735078 0.669287 1.909306 1.843515 2.431854 2.366063 3.745246 3.679455 5.215323 5.149532
492 0.068121 0.727868 0.659747 1.90001 1.831889 2.417185 2.349064 3.697327 3.629206 5.170391 5.10227
492.5 0.071863 0.72077 0.648907 1.892082 1.820219 2.402916 2.331053 3.653334 3.581471 5.132563 5.0607
493 0.075672 0.712789 0.637117 1.885303 1.809631 2.379989 2.304317 3.611121 3.535449 5.103104 5.027432
493.5 0.079394 0.704316 0.624922 1.881637 1.802243 2.349144 2.26975 3.584452 3.505058 5.069952 4.990558
494 0.080268 0.695391 0.615123 1.874674 1.794406 2.316365 2.236097 3.564069 3.483801 5.022875 4.942607
494.5 0.079475 0.686384 0.606909 1.864988 1.785513 2.283875 2.2044 3.545234 3.465759 4.971731 4.892256
495 0.078245 0.679397 0.601152 1.847599 1.769354 2.25221 2.173965 3.532038 3.453793 4.920075 4.84183
495.5 0.076277 0.675597 0.59932 1.826918 1.750641 2.220414 2.144137 3.514622 3.438345 4.869704 4.793427
496 0.072367 0.670359 0.597992 1.804214 1.731847 2.194253 2.121886 3.48829 3.415923 4.824022 4.751655
496.5 0.067996 0.664834 0.596838 1.783339 1.715343 2.17973 2.111734 3.457019 3.389023 4.782542 4.714546
497 0.063936 0.661928 0.597992 1.763531 1.699595 2.175062 2.111126 3.422583 3.358647 4.748275 4.684339
497.5 0.06117 0.661056 0.599886 1.745632 1.684462 2.173006 2.111836 3.382648 3.321478 4.718746 4.657576
498 0.059845 0.661081 0.601236 1.729174 1.669329 2.172541 2.112696 3.352517 3.292672 4.685534 4.625689
498.5 0.058976 0.661538 0.602562 1.712899 1.653923 2.175151 2.116175 3.330111 3.271135 4.637928 4.578952
499 0.058475 0.661417 0.602942 1.694747 1.636272 2.180739 2.122264 3.320443 3.261968 4.576885 4.51841
499.5 0.059934 0.663243 0.603309 1.673112 1.613178 2.185242 2.125308 3.325691 3.265757 4.508523 4.448589
500 0.063 0.668 0.605 1.648 1.585 2.185 2.122 3.338 3.275 4.437 4.374

Figure 2: Blank Corrected Lysozyme Standard Fluorescence by Wavelength

Description of image

Then, the areas underneath the fluorescence emission curve in Figure 2 were integrated using the trapezoid method.

    1. The trapezoid method involves calculating the trapezoidal area incrementally and summing all those areas through the formula (B2+B1)/2*(A2-A1) where A1 is the first wavelength (x-axis) data point and B1 is the first emission intensity (y-axis) data point. Note that the last trapezoid area is not included in the summation as the calculation is skewed by the absence of following data that contributes to the calculation. These areas are shown in Table 4 below.

Table 4: Integrated Lysozyme Fluorescence Spectra by Sample Concentration

Concentration (uM) Integrated Emission Intensity
0.965 4968.558802
1.93 9575.247763
3.86 16960.03477
7.225 26108.92364
15.445 35711.23678

Figure 3: Lysozyme Integrated Standard Fluorescence Intensity by Standard Concentration

Description of image

4) Protease (alpha chymotrypsin) samples were prepped for future experiments with the following procedure (x15 tubes)

    1. Mass was recorded 1 eppendorf tube and then balanced tared
    2. roughly 1 mg of alpha chymotrypsin was added and mass recorded
    3. tube was labeled with protease and concentration after future addition of 1mL water
    4. samples placed in Group 4 (JNB) labeled freezer box and stored for later use

Table 5: Alpha Chymotrypsin Prepped Sample Data

Eppendorf Tube Mass (g) Mass of protease (g) Final Molarity in 1mL H2O
1.015 0.00137 53.515625
1.01348 0.00131 51.171875
1.0179 0.00105 41.015625
1.02954 0.00135 52.734375
1.0228 0.00102 39.84375
1.02048 0.0013 50.78125
1.01945 0.00111 43.359375
1.02723 0.00099 38.671875
1.02951 0.00105 41.015625
1.01517 0.00108 42.1875
1.02954 0.00094 36.71875
1.03211 0.00104 40.625
1.0192 0.00111 43.359375
1.02781 0.00121 47.265625

Matt Hartings This is some enthusiasm here!! You really don't need to put the raw data into your lab notebook pages. Just the graphs should be fine. For your A vs concentration graph, please just show your data in points. Keep the fit as a line. Also what is your molar absorptivity in M-1 cm-1?

Data


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