User:Nicole Bonan/Notebook/Chem 571 Lab Notebook/2015/09/09: Difference between revisions

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[[Image:20150909 bonan intensity conc.png|thumb|center|700px]]
[[Image:20150909 bonan intensity conc.png|thumb|center|700px]]
[[User:Matt Hartings|Matt Hartings]] Lots of data here! You don't have to use the raw data for your spectra. Just show your graphs. In your A vs conc graph, show your data as points only. Keep the line from the fit. What is your measured molar absorptivity M<sup>-1</sup> cm<sup>-1</sup>?


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Revision as of 20:03, 22 September 2015

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Objective

The purpose of today's work is to:

  1. Make stock solutions of lysozyme and determine its molar absorptivity using the UV-Vis and Fluorescence Spectrophotometers
  2. Determine how protein concentration affects fluorescence intensity
  3. Prep a sample of our protease, alpha-chymotrypsin, for use in future experiments

A full description of today's objectives and protocols are in Dr. Hartings's lab notebook.

Procedure

  1. Frist, we made our stock solutions of lysozyme. We made solutions of 50.88µM, 15.445µM, 7.7225µM, 3.86µM, 1.93µM, and 0.96µM lysozyme in HPLC water as follows:
    1. For the 50.88µM solution:
      1. We measured out 7.28mg of the lysozyme on the milligram balance using a weigh boat
      2. We used HPLC water to rinse the lysozyme into a clean 10mL volumetric flask
      3. We filled the volumetric flask to the 10mL line using HPLC water
      4. We transferred the sample into a new 15mL falcon tube
    2. For the remaining solutions:
      1. We weighed out 4.42mg of the lysozyme in a weigh boat using the milligram balance and diluted the lysozyme in HPLC water, using a volumetric flask, to a total volume of 10mL. This solution had a molarity of 30.89µM.
      2. For our first stock solution, we pipetted 5mL of the 30.89µM lysozyme solution into a new 15mL falcon tube and then pipetted 5mL of HPLC into the tube to bring the final molarity to 15.445µM. We labeled the tube in green tape with the concentration.
      3. For our second stock solution, we pipetted 5mL of the 15.445µM solution into a new 15mL falcon tube and then pipetted 5mL of HPLC into the tube to bring the final molarity to 7.7225µM. We labeled the tube in green tape with the concentration.
      4. We continued doing serial dilutions in this way to make three more samples, one of each with a molarity of 3.86µM, 1.93µM, and 0.96µM.
  2. Then, we prepped our stock samples of our protease, alpha-chymotrypsin.
    1. First, we weighed the mass of the 1.5mL Eppindorf tube that we were going to use to store our stock of alpha-chymotrypsin.
    2. Next, we added approximately 1mg of dry alpha-chymotrypsin to the Eppindorf tube.
    3. We then closed the tube and labeled it with the following:
      1. The date (20150909)
      2. The molarity that the stock of alpha-chemotrypsin would be upon addition of 1mL HPLC water
      3. Alpha-chymotrypsin
      4. Our group's initials (BJN)
    4. We did this 15 times. All tubes were stored in a cardboard cryo box with our initials on it in the freezer. A table that shows the mass of the Eppindorf tube, the mass of the alpha-chymotrypsin, and the molarity that the stock would be upon addition of 1mL water is in the Data and Measurements section.
  3. After that, we took absorbance data for each of our lysozyme samples using UV-Vis Spectroscopy.

Data and Measurements

alpha-Chymotrpysin Standards

Table 1: Measurements for Each of the 15 alpha-Chymotrpysin Standards: Mass of Eppindorf Tube (g), Mass of alpha-Chymotrypsin (g), and Molarity of alpha-Chymotrypsin in 1mL HPLC Water (M)


Mass of Eppindorf Tube (g) Mass of alpha-Chymotrypsin (g) Molarity (µM)
1.1017 0.00122 47.656
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

UV-Vis

Table 2: Raw UV-Vis Data for Blank (HPLC Water) and All Lysozyme Standards for Wavelengths 200-800nm


Wavelength (nm) Absorbance of Blank (HPLC Water) (0M) Absorbance of 15.445 µM Standard Absorbance of 7.225µM Standard Absorbance of 3.86µM Standard Absorbance of 1.93µM Standard Absorbance of 0.965µM Standard
200 0.091 5 4.798 2.556 1.305 0.686
201 0.084 5 4.798 2.356 1.203 0.634
202 0.08 5 4.023 2.165 1.104 0.582
203 0.075 5 3.898 1.987 1.016 0.536
204 0.071 5 3.573 1.832 0.94 0.495
205 0.068 5 3.273 1.698 0.874 0.459
206 0.065 5 3.056 1.58 0.817 0.428
207 0.062 5 2.846 1.474 0.763 0.4
208 0.06 5 2.67 1.384 0.717 0.376
209 0.057 5 2.532 1.306 0.679 0.355
210 0.056 5 2.409 1.246 0.647 0.339
211 0.054 5 2.306 1.192 0.62 0.325
212 0.052 4.498 2.219 1.148 0.597 0.313
213 0.052 4.498 2.149 1.111 0.578 0.304
214 0.051 4.498 2.097 1.084 0.564 0.297
215 0.05 4.198 2.056 1.062 0.552 0.29
216 0.049 4.198 2.023 1.044 0.544 0.286
217 0.048 4.102 1.996 1.031 0.536 0.283
218 0.047 4.102 1.975 1.018 0.529 0.28
219 0.046 3.956 1.95 1.006 0.522 0.276
220 0.046 3.848 1.922 0.99 0.515 0.273
221 0.046 3.761 1.885 0.972 0.505 0.268
222 0.045 3.656 1.835 0.947 0.492 0.262
223 0.045 3.502 1.777 0.917 0.477 0.254
224 0.045 3.372 1.704 0.882 0.459 0.246
225 0.044 3.203 1.626 0.843 0.439 0.236
226 0.043 3.005 1.531 0.795 0.416 0.224
227 0.043 2.784 1.429 0.744 0.39 0.211
228 0.042 2.556 1.315 0.686 0.362 0.197
229 0.042 2.3 1.189 0.622 0.331 0.181
230 0.041 2.038 1.059 0.557 0.298 0.164
231 0.04 1.776 0.926 0.489 0.265 0.147
232 0.039 1.539 0.806 0.427 0.235 0.131
233 0.038 1.325 0.694 0.37 0.206 0.117
234 0.038 1.13 0.593 0.318 0.18 0.104
235 0.037 0.972 0.512 0.276 0.159 0.093
236 0.036 0.842 0.443 0.241 0.141 0.083
237 0.036 0.738 0.39 0.213 0.127 0.077
238 0.035 0.65 0.345 0.191 0.115 0.071
239 0.035 0.574 0.306 0.171 0.105 0.065
240 0.035 0.513 0.275 0.155 0.097 0.061
241 0.034 0.461 0.249 0.141 0.09 0.058
242 0.034 0.416 0.227 0.13 0.085 0.055
243 0.033 0.381 0.209 0.12 0.08 0.053
244 0.033 0.351 0.194 0.113 0.076 0.05
245 0.033 0.328 0.182 0.107 0.073 0.049
246 0.032 0.31 0.172 0.102 0.07 0.047
247 0.032 0.295 0.165 0.097 0.068 0.046
248 0.032 0.285 0.159 0.095 0.067 0.045
249 0.032 0.278 0.155 0.093 0.066 0.045
250 0.032 0.274 0.153 0.091 0.065 0.044
251 0.031 0.273 0.152 0.091 0.064 0.044
252 0.031 0.274 0.153 0.091 0.064 0.043
253 0.031 0.277 0.154 0.091 0.064 0.043
254 0.03 0.282 0.157 0.093 0.065 0.044
255 0.03 0.289 0.161 0.095 0.065 0.044
256 0.03 0.298 0.165 0.097 0.066 0.044
257 0.03 0.309 0.17 0.1 0.068 0.045
258 0.03 0.32 0.176 0.102 0.068 0.046
259 0.03 0.332 0.182 0.105 0.07 0.046
260 0.03 0.344 0.188 0.108 0.071 0.046
261 0.03 0.357 0.195 0.111 0.072 0.047
262 0.03 0.371 0.202 0.115 0.074 0.048
263 0.029 0.386 0.21 0.118 0.076 0.049
264 0.029 0.401 0.216 0.122 0.077 0.049
265 0.029 0.415 0.224 0.125 0.079 0.05
266 0.029 0.43 0.231 0.129 0.081 0.051
267 0.029 0.445 0.239 0.133 0.082 0.051
268 0.029 0.461 0.246 0.136 0.084 0.052
269 0.029 0.474 0.253 0.14 0.086 0.053
270 0.029 0.488 0.26 0.143 0.088 0.054
271 0.028 0.499 0.265 0.146 0.089 0.054
272 0.028 0.509 0.27 0.148 0.089 0.054
273 0.029 0.517 0.275 0.15 0.09 0.055
274 0.028 0.524 0.277 0.151 0.091 0.056
275 0.028 0.528 0.279 0.152 0.091 0.056
276 0.028 0.53 0.28 0.152 0.091 0.055
277 0.028 0.531 0.281 0.153 0.091 0.055
278 0.028 0.535 0.283 0.154 0.092 0.056
279 0.028 0.539 0.285 0.155 0.093 0.056
280 0.028 0.543 0.287 0.156 0.093 0.056
281 0.027 0.544 0.287 0.156 0.093 0.056
282 0.027 0.541 0.286 0.155 0.092 0.056
283 0.028 0.533 0.282 0.153 0.091 0.055
284 0.028 0.518 0.274 0.149 0.089 0.054
285 0.027 0.495 0.263 0.143 0.086 0.053
286 0.027 0.469 0.25 0.136 0.083 0.051
287 0.027 0.443 0.238 0.131 0.08 0.05
288 0.028 0.428 0.23 0.127 0.079 0.049
289 0.027 0.424 0.228 0.126 0.079 0.049
290 0.027 0.422 0.227 0.125 0.078 0.048
291 0.027 0.411 0.222 0.123 0.077 0.048
292 0.027 0.383 0.207 0.115 0.073 0.046
293 0.027 0.337 0.184 0.104 0.067 0.043
294 0.027 0.288 0.159 0.092 0.061 0.04
295 0.027 0.241 0.135 0.079 0.056 0.037
296 0.027 0.202 0.115 0.069 0.051 0.034
297 0.027 0.169 0.098 0.061 0.047 0.032
298 0.027 0.142 0.084 0.054 0.043 0.031
299 0.026 0.118 0.073 0.048 0.041 0.029
300 0.026 0.099 0.063 0.044 0.039 0.028
301 0.027 0.084 0.056 0.04 0.037 0.028
302 0.027 0.07 0.049 0.037 0.035 0.027
303 0.027 0.061 0.045 0.035 0.034 0.026
304 0.027 0.054 0.041 0.033 0.033 0.026
305 0.027 0.049 0.038 0.031 0.033 0.026
306 0.026 0.044 0.036 0.03 0.031 0.025
307 0.027 0.041 0.034 0.03 0.031 0.025
308 0.027 0.039 0.033 0.029 0.031 0.025
309 0.027 0.038 0.032 0.028 0.03 0.024
310 0.027 0.036 0.032 0.028 0.031 0.024
311 0.027 0.035 0.032 0.028 0.03 0.024
312 0.027 0.035 0.031 0.028 0.03 0.024
313 0.027 0.035 0.031 0.028 0.03 0.024
314 0.027 0.034 0.03 0.028 0.03 0.024
315 0.027 0.034 0.03 0.028 0.03 0.024
316 0.027 0.034 0.03 0.028 0.03 0.025
317 0.027 0.034 0.03 0.028 0.03 0.024
318 0.028 0.034 0.031 0.028 0.03 0.024
319 0.027 0.034 0.03 0.028 0.03 0.025
320 0.026 0.033 0.029 0.027 0.029 0.024
321 0.026 0.033 0.029 0.027 0.029 0.023
322 0.026 0.032 0.029 0.026 0.029 0.024
323 0.026 0.033 0.03 0.027 0.029 0.024
324 0.027 0.033 0.029 0.027 0.03 0.024
325 0.027 0.032 0.029 0.027 0.029 0.024
326 0.026 0.032 0.029 0.027 0.029 0.024
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328 0.027 0.033 0.029 0.028 0.029 0.024
329 0.026 0.032 0.029 0.028 0.029 0.024
330 0.027 0.032 0.029 0.027 0.029 0.024
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333 0.027 0.032 0.029 0.027 0.03 0.024
334 0.026 0.031 0.029 0.027 0.029 0.024
335 0.026 0.031 0.029 0.027 0.029 0.024
336 0.026 0.032 0.029 0.027 0.029 0.024
337 0.026 0.031 0.029 0.027 0.029 0.024
338 0.026 0.032 0.028 0.027 0.029 0.023
339 0.026 0.031 0.029 0.027 0.028 0.023
340 0.027 0.031 0.029 0.027 0.029 0.024
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378 0.027 0.029 0.027 0.027 0.027 0.023
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381 0.027 0.03 0.027 0.028 0.027 0.024
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387 0.027 0.029 0.027 0.027 0.027 0.023
388 0.026 0.029 0.027 0.028 0.027 0.023
389 0.027 0.03 0.027 0.028 0.027 0.023
390 0.027 0.029 0.027 0.028 0.027 0.024
391 0.027 0.029 0.027 0.028 0.027 0.024
392 0.027 0.029 0.028 0.028 0.027 0.024
393 0.027 0.029 0.027 0.028 0.027 0.024
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395 0.027 0.029 0.026 0.028 0.027 0.023
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399 0.028 0.029 0.027 0.029 0.027 0.024
400 0.028 0.029 0.028 0.029 0.027 0.024
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402 0.028 0.03 0.027 0.03 0.027 0.025
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416 0.03 0.029 0.027 0.031 0.028 0.026
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425 0.029 0.029 0.027 0.03 0.027 0.025
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428 0.028 0.029 0.027 0.029 0.027 0.025
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441 0.028 0.028 0.027 0.028 0.027 0.025
442 0.028 0.028 0.026 0.029 0.027 0.024
443 0.027 0.028 0.026 0.028 0.027 0.024
444 0.027 0.028 0.026 0.028 0.026 0.023
445 0.027 0.028 0.026 0.029 0.027 0.024
446 0.028 0.028 0.027 0.029 0.027 0.024
447 0.027 0.028 0.026 0.028 0.027 0.024
448 0.028 0.028 0.026 0.029 0.027 0.024
449 0.028 0.028 0.026 0.029 0.027 0.024
450 0.028 0.029 0.026 0.029 0.026 0.025
451 0.028 0.028 0.027 0.029 0.027 0.024
452 0.029 0.028 0.027 0.029 0.027 0.025
453 0.029 0.028 0.027 0.03 0.027 0.025
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497 0.032 0.028 0.027 0.032 0.027 0.027
498 0.032 0.029 0.027 0.033 0.027 0.027
499 0.032 0.029 0.027 0.033 0.027 0.027
500 0.032 0.028 0.027 0.033 0.027 0.027
501 0.032 0.028 0.027 0.032 0.027 0.027
502 0.032 0.028 0.027 0.032 0.027 0.027
503 0.031 0.029 0.027 0.031 0.027 0.026
504 0.031 0.029 0.027 0.031 0.027 0.026
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508 0.03 0.028 0.027 0.03 0.027 0.026
509 0.029 0.028 0.027 0.03 0.027 0.026
510 0.03 0.028 0.027 0.03 0.027 0.026
511 0.029 0.028 0.026 0.03 0.027 0.026
512 0.029 0.028 0.027 0.03 0.027 0.026
513 0.029 0.028 0.027 0.03 0.027 0.026
514 0.029 0.028 0.027 0.03 0.027 0.026
515 0.03 0.028 0.027 0.03 0.027 0.026
516 0.029 0.028 0.027 0.03 0.027 0.026
517 0.029 0.028 0.027 0.03 0.027 0.026
518 0.03 0.028 0.027 0.03 0.027 0.026
519 0.029 0.028 0.027 0.03 0.027 0.026
520 0.029 0.028 0.027 0.03 0.027 0.026
521 0.029 0.028 0.027 0.03 0.027 0.026
522 0.029 0.028 0.027 0.03 0.027 0.026
523 0.03 0.028 0.027 0.03 0.027 0.026
524 0.029 0.028 0.027 0.03 0.027 0.026
525 0.029 0.028 0.027 0.03 0.027 0.026
526 0.029 0.028 0.027 0.03 0.027 0.026
527 0.03 0.028 0.027 0.031 0.027 0.026
528 0.03 0.028 0.027 0.031 0.027 0.027
529 0.03 0.028 0.027 0.031 0.028 0.027
530 0.03 0.028 0.027 0.031 0.027 0.027
531 0.03 0.029 0.027 0.031 0.027 0.027
532 0.031 0.029 0.027 0.031 0.027 0.027
533 0.031 0.028 0.027 0.032 0.028 0.027
534 0.032 0.029 0.027 0.032 0.028 0.028
535 0.031 0.028 0.027 0.032 0.028 0.027
536 0.031 0.028 0.027 0.032 0.028 0.027
537 0.031 0.029 0.028 0.029 0.027 0.027
538 0.031 0.028 0.028 0.031 0.027 0.027
539 0.032 0.029 0.027 0.033 0.028 0.028
540 0.032 0.028 0.028 0.033 0.028 0.028
541 0.032 0.028 0.027 0.033 0.028 0.028
542 0.031 0.029 0.027 0.032 0.028 0.027
543 0.031 0.028 0.027 0.032 0.027 0.027
544 0.031 0.028 0.027 0.032 0.027 0.027
545 0.031 0.028 0.027 0.033 0.028 0.027
546 0.032 0.028 0.028 0.032 0.028 0.027
547 0.031 0.029 0.028 0.032 0.028 0.027
548 0.031 0.028 0.027 0.032 0.027 0.027
549 0.031 0.028 0.027 0.032 0.027 0.027
550 0.031 0.028 0.027 0.032 0.027 0.027
551 0.031 0.028 0.027 0.032 0.027 0.027
552 0.031 0.028 0.028 0.032 0.028 0.027
553 0.031 0.028 0.027 0.032 0.027 0.027
554 0.031 0.028 0.027 0.032 0.027 0.027
555 0.03 0.028 0.027 0.032 0.027 0.027
556 0.03 0.028 0.027 0.031 0.028 0.027
557 0.03 0.028 0.027 0.031 0.027 0.027
558 0.03 0.028 0.027 0.032 0.027 0.027
559 0.03 0.028 0.027 0.031 0.027 0.027
560 0.03 0.028 0.027 0.032 0.027 0.027
561 0.03 0.028 0.027 0.031 0.027 0.026
562 0.03 0.028 0.027 0.031 0.027 0.026
563 0.03 0.028 0.027 0.031 0.027 0.026
564 0.03 0.028 0.027 0.031 0.027 0.027
565 0.03 0.028 0.027 0.031 0.027 0.026
566 0.03 0.028 0.027 0.031 0.027 0.026
567 0.03 0.028 0.027 0.031 0.027 0.026
568 0.03 0.029 0.027 0.031 0.027 0.027
569 0.03 0.028 0.027 0.031 0.027 0.027
570 0.03 0.028 0.027 0.031 0.027 0.026
571 0.03 0.028 0.027 0.031 0.027 0.026
572 0.03 0.028 0.027 0.031 0.027 0.026
573 0.03 0.028 0.027 0.031 0.027 0.026
574 0.03 0.028 0.027 0.031 0.027 0.026
575 0.03 0.028 0.027 0.031 0.027 0.026
576 0.03 0.028 0.027 0.031 0.027 0.026
577 0.029 0.028 0.027 0.031 0.027 0.026
578 0.03 0.028 0.027 0.031 0.027 0.026
579 0.03 0.028 0.027 0.031 0.027 0.026
580 0.03 0.028 0.027 0.031 0.027 0.026
581 0.03 0.028 0.027 0.031 0.027 0.026
582 0.03 0.028 0.027 0.031 0.027 0.027
583 0.03 0.028 0.027 0.031 0.027 0.027
584 0.03 0.028 0.027 0.032 0.027 0.027
585 0.03 0.028 0.027 0.031 0.027 0.026
586 0.03 0.029 0.027 0.032 0.027 0.027
587 0.03 0.029 0.028 0.032 0.028 0.027
588 0.03 0.029 0.027 0.032 0.028 0.027
589 0.03 0.028 0.027 0.032 0.027 0.027
590 0.03 0.029 0.028 0.032 0.027 0.027
591 0.03 0.029 0.027 0.032 0.027 0.027
592 0.03 0.029 0.028 0.032 0.028 0.027
593 0.03 0.029 0.028 0.032 0.028 0.027
594 0.03 0.029 0.028 0.032 0.028 0.027
595 0.03 0.029 0.028 0.032 0.028 0.027
596 0.03 0.029 0.028 0.032 0.028 0.027
597 0.03 0.029 0.028 0.032 0.028 0.027
598 0.03 0.029 0.028 0.032 0.028 0.027
599 0.03 0.029 0.028 0.032 0.028 0.027
600 0.031 0.029 0.028 0.032 0.028 0.027
601 0.031 0.029 0.028 0.032 0.028 0.027
602 0.031 0.029 0.028 0.033 0.028 0.027
603 0.031 0.029 0.028 0.033 0.028 0.027
604 0.031 0.03 0.029 0.033 0.028 0.028
605 0.031 0.03 0.028 0.033 0.028 0.028
606 0.031 0.03 0.028 0.033 0.028 0.027
607 0.031 0.029 0.028 0.033 0.028 0.027
608 0.031 0.029 0.028 0.033 0.028 0.027
609 0.031 0.03 0.029 0.033 0.028 0.028
610 0.031 0.03 0.028 0.033 0.028 0.028
611 0.031 0.03 0.028 0.033 0.028 0.027
612 0.031 0.029 0.028 0.033 0.028 0.028
613 0.031 0.03 0.029 0.033 0.028 0.028
614 0.031 0.029 0.028 0.033 0.028 0.028
615 0.031 0.03 0.028 0.033 0.028 0.028
616 0.031 0.03 0.028 0.033 0.029 0.028
617 0.031 0.03 0.029 0.033 0.028 0.028
618 0.031 0.03 0.028 0.033 0.028 0.028
619 0.031 0.029 0.028 0.033 0.028 0.028
620 0.031 0.03 0.028 0.033 0.028 0.028
621 0.031 0.03 0.028 0.033 0.028 0.027
622 0.031 0.03 0.029 0.033 0.028 0.028
623 0.031 0.03 0.028 0.033 0.029 0.028
624 0.031 0.03 0.028 0.033 0.028 0.028
625 0.031 0.03 0.029 0.033 0.028 0.028
626 0.031 0.03 0.029 0.033 0.029 0.028
627 0.031 0.03 0.028 0.033 0.028 0.028
628 0.031 0.03 0.028 0.033 0.029 0.028
629 0.031 0.03 0.029 0.033 0.028 0.028
630 0.031 0.03 0.029 0.033 0.029 0.028
631 0.032 0.03 0.029 0.033 0.028 0.028
632 0.032 0.03 0.029 0.034 0.029 0.028
633 0.032 0.03 0.029 0.034 0.029 0.028
634 0.032 0.03 0.029 0.034 0.029 0.028
635 0.032 0.03 0.029 0.033 0.029 0.028
636 0.032 0.03 0.029 0.033 0.029 0.028
637 0.032 0.03 0.029 0.033 0.029 0.028
638 0.032 0.03 0.029 0.033 0.029 0.028
639 0.032 0.03 0.029 0.034 0.029 0.028
640 0.031 0.03 0.029 0.034 0.029 0.028
641 0.032 0.03 0.029 0.033 0.029 0.028
642 0.031 0.03 0.029 0.033 0.029 0.028
643 0.031 0.03 0.029 0.033 0.029 0.028
644 0.032 0.03 0.029 0.034 0.029 0.028
645 0.032 0.03 0.029 0.033 0.029 0.028
646 0.032 0.03 0.029 0.033 0.029 0.028
647 0.031 0.03 0.029 0.033 0.029 0.028
648 0.031 0.03 0.029 0.033 0.029 0.028
649 0.032 0.03 0.029 0.034 0.029 0.028
650 0.032 0.03 0.029 0.034 0.029 0.028
651 0.032 0.03 0.029 0.034 0.029 0.028
652 0.032 0.03 0.029 0.034 0.029 0.028
653 0.032 0.03 0.029 0.034 0.029 0.028
654 0.032 0.03 0.029 0.034 0.029 0.028
655 0.032 0.03 0.029 0.034 0.029 0.028
656 0.032 0.031 0.029 0.034 0.029 0.028
657 0.032 0.031 0.029 0.034 0.029 0.028
658 0.032 0.031 0.029 0.034 0.029 0.028
659 0.032 0.03 0.029 0.034 0.029 0.028
660 0.032 0.031 0.029 0.034 0.029 0.028
661 0.032 0.031 0.03 0.034 0.029 0.028
662 0.032 0.03 0.03 0.034 0.029 0.029
663 0.032 0.03 0.029 0.034 0.029 0.028
664 0.032 0.031 0.03 0.034 0.029 0.028
665 0.032 0.03 0.03 0.034 0.029 0.028
666 0.032 0.03 0.029 0.034 0.029 0.028
667 0.032 0.03 0.03 0.034 0.029 0.028
668 0.032 0.031 0.03 0.034 0.03 0.028
669 0.032 0.031 0.03 0.034 0.03 0.028
670 0.032 0.03 0.03 0.034 0.03 0.028
671 0.032 0.03 0.03 0.033 0.029 0.028
672 0.032 0.03 0.03 0.034 0.029 0.028
673 0.032 0.031 0.03 0.034 0.029 0.028
674 0.032 0.03 0.03 0.033 0.029 0.028
675 0.032 0.03 0.03 0.034 0.029 0.028
676 0.032 0.031 0.03 0.034 0.03 0.028
677 0.032 0.031 0.03 0.034 0.03 0.028
678 0.032 0.03 0.03 0.034 0.029 0.028
679 0.032 0.031 0.03 0.034 0.029 0.028
680 0.032 0.031 0.03 0.034 0.029 0.028
681 0.032 0.03 0.03 0.034 0.03 0.028
682 0.032 0.031 0.03 0.033 0.029 0.028
683 0.032 0.031 0.03 0.034 0.029 0.028
684 0.032 0.03 0.03 0.034 0.029 0.028
685 0.032 0.031 0.03 0.034 0.03 0.029
686 0.032 0.03 0.03 0.034 0.03 0.029
687 0.032 0.031 0.03 0.034 0.03 0.029
688 0.032 0.031 0.03 0.034 0.03 0.029
689 0.032 0.031 0.03 0.034 0.03 0.028
690 0.032 0.031 0.03 0.034 0.029 0.028
691 0.032 0.031 0.03 0.034 0.03 0.029
692 0.032 0.031 0.03 0.033 0.03 0.029
693 0.031 0.031 0.03 0.034 0.03 0.028
694 0.032 0.031 0.03 0.034 0.03 0.028
695 0.032 0.031 0.03 0.033 0.03 0.029
696 0.032 0.031 0.03 0.034 0.03 0.029
697 0.032 0.031 0.031 0.034 0.03 0.028
698 0.032 0.031 0.03 0.034 0.03 0.029
699 0.031 0.031 0.03 0.033 0.03 0.028
700 0.032 0.031 0.03 0.033 0.03 0.028
701 0.032 0.031 0.03 0.034 0.03 0.028
702 0.031 0.031 0.031 0.034 0.03 0.028
703 0.032 0.031 0.031 0.034 0.03 0.028
704 0.032 0.031 0.031 0.034 0.031 0.029
705 0.032 0.031 0.031 0.034 0.03 0.029
706 0.032 0.031 0.031 0.035 0.031 0.029
707 0.032 0.032 0.031 0.034 0.03 0.029
708 0.032 0.032 0.031 0.034 0.031 0.029
709 0.032 0.032 0.032 0.035 0.031 0.029
710 0.033 0.032 0.032 0.035 0.031 0.03
711 0.032 0.032 0.032 0.035 0.031 0.029
712 0.032 0.032 0.032 0.034 0.031 0.029
713 0.033 0.032 0.032 0.035 0.032 0.03
714 0.033 0.033 0.032 0.035 0.032 0.03
715 0.033 0.033 0.032 0.035 0.032 0.03
716 0.034 0.033 0.033 0.035 0.032 0.031
717 0.034 0.033 0.033 0.036 0.032 0.031
718 0.034 0.033 0.033 0.036 0.033 0.031
719 0.034 0.034 0.033 0.036 0.033 0.031
720 0.035 0.034 0.034 0.037 0.033 0.031
721 0.035 0.034 0.034 0.037 0.033 0.032
722 0.035 0.034 0.034 0.037 0.034 0.032
723 0.035 0.035 0.034 0.037 0.034 0.032
724 0.036 0.035 0.035 0.037 0.034 0.032
725 0.036 0.035 0.035 0.038 0.034 0.033
726 0.036 0.035 0.035 0.039 0.035 0.033
727 0.037 0.036 0.036 0.039 0.035 0.034
728 0.038 0.036 0.037 0.04 0.035 0.034
729 0.038 0.037 0.037 0.041 0.036 0.035
730 0.038 0.037 0.037 0.041 0.036 0.035
731 0.039 0.037 0.038 0.041 0.036 0.035
732 0.039 0.038 0.038 0.042 0.037 0.036
733 0.04 0.039 0.039 0.043 0.038 0.036
734 0.04 0.039 0.039 0.043 0.038 0.037
735 0.041 0.039 0.039 0.043 0.039 0.037
736 0.041 0.04 0.04 0.044 0.039 0.038
737 0.042 0.04 0.04 0.044 0.04 0.038
738 0.041 0.041 0.041 0.044 0.04 0.038
739 0.042 0.041 0.041 0.044 0.04 0.038
740 0.042 0.041 0.041 0.045 0.04 0.038
741 0.042 0.041 0.041 0.044 0.04 0.038
742 0.042 0.041 0.041 0.045 0.04 0.038
743 0.042 0.041 0.041 0.045 0.04 0.039
744 0.042 0.041 0.041 0.045 0.04 0.038
745 0.042 0.041 0.041 0.045 0.04 0.038
746 0.042 0.041 0.041 0.044 0.04 0.039
747 0.042 0.041 0.041 0.045 0.04 0.039
748 0.042 0.041 0.041 0.045 0.04 0.039
749 0.042 0.041 0.041 0.045 0.041 0.038
750 0.043 0.041 0.041 0.045 0.04 0.039
751 0.042 0.041 0.041 0.045 0.04 0.039
752 0.042 0.041 0.041 0.044 0.04 0.039
753 0.043 0.041 0.042 0.046 0.041 0.039
754 0.042 0.041 0.042 0.045 0.04 0.039
755 0.042 0.041 0.041 0.046 0.04 0.039
756 0.042 0.041 0.041 0.046 0.04 0.039
757 0.042 0.041 0.041 0.045 0.04 0.039
758 0.042 0.042 0.042 0.045 0.04 0.039
759 0.042 0.041 0.042 0.046 0.041 0.039
760 0.043 0.041 0.041 0.046 0.04 0.039
761 0.042 0.041 0.041 0.046 0.04 0.039
762 0.043 0.041 0.042 0.045 0.04 0.039
763 0.042 0.041 0.041 0.045 0.04 0.039
764 0.042 0.041 0.041 0.046 0.04 0.039
765 0.043 0.041 0.042 0.046 0.041 0.039
766 0.042 0.041 0.042 0.046 0.04 0.039
767 0.042 0.041 0.041 0.045 0.04 0.039
768 0.043 0.041 0.042 0.046 0.04 0.039
769 0.043 0.041 0.041 0.047 0.04 0.039
770 0.042 0.041 0.041 0.046 0.04 0.039
771 0.043 0.041 0.042 0.046 0.04 0.038
772 0.042 0.04 0.041 0.046 0.039 0.039
773 0.042 0.04 0.041 0.046 0.039 0.039
774 0.042 0.04 0.041 0.047 0.04 0.038
775 0.042 0.04 0.041 0.046 0.039 0.038
776 0.042 0.04 0.041 0.045 0.039 0.038
777 0.043 0.04 0.041 0.047 0.04 0.039
778 0.042 0.04 0.041 0.046 0.039 0.039
779 0.042 0.04 0.041 0.046 0.039 0.038
780 0.042 0.04 0.04 0.046 0.039 0.038
781 0.042 0.039 0.041 0.045 0.039 0.038
782 0.041 0.04 0.04 0.045 0.038 0.037
783 0.042 0.04 0.04 0.045 0.038 0.038
784 0.041 0.039 0.04 0.045 0.038 0.038
785 0.041 0.039 0.04 0.046 0.038 0.038
786 0.041 0.039 0.039 0.045 0.038 0.037
787 0.041 0.039 0.039 0.046 0.038 0.037
788 0.041 0.04 0.04 0.046 0.038 0.038
789 0.041 0.039 0.039 0.045 0.038 0.038
790 0.041 0.038 0.039 0.046 0.038 0.037
791 0.041 0.038 0.039 0.046 0.038 0.037
792 0.041 0.039 0.039 0.046 0.038 0.038
793 0.04 0.038 0.039 0.045 0.037 0.037
794 0.04 0.038 0.039 0.044 0.037 0.037
795 0.039 0.037 0.038 0.044 0.037 0.036
796 0.04 0.038 0.039 0.043 0.037 0.036
797 0.04 0.038 0.038 0.045 0.037 0.036
798 0.039 0.038 0.038 0.043 0.037 0.036
799 0.04 0.038 0.038 0.045 0.037 0.036
800 0.04 0.038 0.038 0.045 0.037 0.036

Fluorescence

Table 3: Raw Fluorescence Data for Blank (HPLC Water) and Lysozyme Standards for Wavelengths 300-500nm


Wavelength (nm) Fluorescence of Blank (HPLC Water) (0M) Fluorescence of 0.965M Standard Fluorescence of 1.93M Standard Fluorescence of 3.86M Standard Fluorescence of 7.225M Standard Fluorescence of 15.445M Standard
300.00 0.241000 1.714000 4.044000 7.223000 10.010000 14.053000
300.50 0.231798 2.033250 4.319538 7.736019 11.304191 15.803222
301.00 0.225261 2.338888 4.614535 8.285435 12.590322 17.547442
301.50 0.223047 2.635071 4.949361 8.903301 13.889913 19.315293
302.00 0.228552 2.924992 5.323778 9.592161 15.225661 21.129509
302.50 0.244522 3.212784 5.739772 10.375192 16.603248 23.008914
303.00 0.267249 3.498136 6.213206 11.250088 18.029729 24.981096
303.50 0.295490 3.782348 6.743837 12.221671 19.515792 27.078760
304.00 0.330513 4.080415 7.320252 13.279723 21.111722 29.280986
304.50 0.372482 4.407780 7.943491 14.416857 22.836812 31.628813
305.00 0.419023 4.760684 8.607499 15.626171 24.688707 34.156151
305.50 0.471697 5.145484 9.310102 16.909374 26.640423 36.849717
306.00 0.528381 5.561600 10.049318 18.272295 28.692961 39.692439
306.50 0.588148 6.018216 10.819654 19.712849 30.882519 42.721680
307.00 0.651476 6.509498 11.627992 21.225987 33.172505 45.961683
307.50 0.715064 7.028985 12.486288 22.815255 35.581102 49.374521
308.00 0.777629 7.568474 13.405930 24.481414 38.077647 52.984636
308.50 0.841886 8.136667 14.385542 26.221722 40.687618 56.748090
309.00 0.910814 8.733959 15.436765 28.039348 43.432797 60.633703
309.50 0.979747 9.348815 16.551743 29.930526 46.336476 64.700986
310.00 1.052985 9.993592 17.723677 31.897695 49.355132 68.941426
310.50 1.128943 10.657105 18.951345 33.931165 52.486879 73.304546
311.00 1.203638 11.353547 20.218586 36.037246 55.762869 77.841888
311.50 1.275243 12.081265 21.516561 38.248507 59.123397 82.575548
312.00 1.341320 12.833839 22.839916 40.548694 62.639971 87.489119
312.50 1.401930 13.600307 24.199256 42.925690 66.286749 92.622357
313.00 1.457897 14.378439 25.590860 45.378449 70.040078 97.957017
313.50 1.509168 15.166029 27.030541 47.930261 73.937197 103.471745
314.00 1.550367 15.956243 28.514118 50.578133 77.983355 109.193128
314.50 1.588591 16.761309 30.036566 53.278026 82.149906 115.083112
315.00 1.622839 17.561858 31.604185 56.004031 86.473877 121.074565
315.50 1.653910 18.368003 33.199677 58.741792 90.905967 127.150642
316.00 1.676645 19.189059 34.802280 61.546736 95.373750 133.292232
316.50 1.688100 20.027096 36.411613 64.416710 99.913260 139.454796
317.00 1.687316 20.874330 38.033591 67.332325 104.517087 145.680434
317.50 1.677851 21.723087 39.664651 70.268426 109.123967 152.002493
318.00 1.659588 22.578469 41.302072 73.241000 113.809483 158.365797
318.50 1.627299 23.447834 42.957462 76.282643 118.490301 164.873876
319.00 1.588279 24.344866 44.643633 79.370905 123.136889 171.524240
319.50 1.540120 25.262456 46.395009 82.476069 127.886727 178.311649
320.00 1.487784 26.172040 48.197345 85.580486 132.708429 185.261878
320.50 1.431515 27.061038 50.031867 88.739111 137.537459 192.141519
321.00 1.370547 27.943139 51.899806 91.962865 142.371398 198.965641
321.50 1.303949 28.825355 53.791944 95.240965 147.229535 205.774010
322.00 1.234534 29.679596 55.680727 98.555997 152.071789 212.529652
322.50 1.163718 30.506110 57.551065 101.807071 156.967597 219.002715
323.00 1.088592 31.321452 59.407955 105.029333 161.811408 225.306517
323.50 1.013374 32.143539 61.250589 108.216203 166.503724 231.494407
324.00 0.935516 32.998722 63.092338 111.295935 171.187025 237.596126
324.50 0.858543 33.878885 64.932341 114.326070 175.905241 243.747562
325.00 0.786717 34.756982 66.764450 117.288911 180.566628 249.734639
325.50 0.721899 35.649098 68.604459 120.226812 185.109834 255.653893
326.00 0.662493 36.549759 70.440135 123.169980 189.636505 261.613026
326.50 0.608481 37.438986 72.235667 126.187625 194.113282 267.782319
327.00 0.561378 38.302427 74.017799 129.204503 198.611000 274.018876
327.50 0.517635 39.144049 75.808014 132.178958 203.068188 280.325295
328.00 0.477328 39.964845 77.564340 135.168428 207.480860 286.816561
328.50 0.440311 40.776506 79.294141 138.126234 211.943094 293.341866
329.00 0.404451 41.594963 81.013649 141.124212 216.595542 299.951529
329.50 0.371818 42.406246 82.745713 144.111031 221.309561 306.357598
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453.00 0.128621 2.455453 5.306440 8.044594 12.653587 17.548163
453.50 0.127061 2.404509 5.213040 7.858088 12.356118 17.170490
454.00 0.124396 2.353308 5.119316 7.688640 12.066343 16.807409
454.50 0.120768 2.302635 5.025524 7.538431 11.796886 16.457915
455.00 0.115057 2.256183 4.932731 7.403724 11.538076 16.127911
455.50 0.109699 2.212005 4.839600 7.272688 11.294062 15.798616
456.00 0.106269 2.165590 4.747361 7.147958 11.069739 15.461571
456.50 0.104884 2.119696 4.656631 7.021148 10.863505 15.119931
457.00 0.104608 2.072950 4.564393 6.892557 10.655410 14.792868
457.50 0.105958 2.025725 4.471174 6.750106 10.443017 14.469215
458.00 0.108230 1.979651 4.392610 6.598314 10.225838 14.156946
458.50 0.111584 1.932622 4.315147 6.440129 10.012332 13.849794
459.00 0.112509 1.888250 4.238628 6.287152 9.803061 13.548789
459.50 0.110564 1.848319 4.165503 6.139908 9.594397 13.266492
460.00 0.107337 1.812232 4.095596 5.998609 9.382381 12.989650
460.50 0.103632 1.780220 4.030624 5.871743 9.185847 12.718943
461.00 0.098064 1.753873 3.973367 5.762399 9.005024 12.448622
461.50 0.091398 1.729105 3.916768 5.664781 8.826698 12.189978
462.00 0.086238 1.703874 3.852803 5.566949 8.649068 11.951732
462.50 0.083283 1.679229 3.792756 5.471155 8.474225 11.727490
463.00 0.084834 1.650765 3.725643 5.369943 8.305142 11.511942
463.50 0.087382 1.620276 3.652324 5.266337 8.137822 11.302113
464.00 0.089726 1.587388 3.574561 5.159191 7.969741 11.099427
464.50 0.092708 1.550840 3.497780 5.049099 7.800986 10.897618
465.00 0.095336 1.512421 3.425784 4.941380 7.642719 10.692595
465.50 0.095976 1.473782 3.365278 4.839634 7.490625 10.483267
466.00 0.093990 1.439567 3.316317 4.745137 7.346521 10.264016
466.50 0.090498 1.411064 3.273919 4.647761 7.220312 10.057934
467.00 0.086796 1.388671 3.232453 4.557805 7.103315 9.859264
467.50 0.084668 1.368226 3.189030 4.475139 6.996225 9.675906
468.00 0.083650 1.353595 3.147103 4.396376 6.890356 9.513829
468.50 0.083877 1.341457 3.100533 4.314938 6.785614 9.369235
469.00 0.085215 1.327460 3.055874 4.238876 6.680655 9.244495
469.50 0.086624 1.310635 3.012067 4.168760 6.581571 9.123980
470.00 0.087442 1.286096 2.972592 4.102112 6.477173 9.005117
470.50 0.087902 1.261538 2.939849 4.044315 6.369455 8.867348
471.00 0.086836 1.237685 2.911629 3.982841 6.265521 8.726090
471.50 0.084699 1.212456 2.876779 3.922584 6.163749 8.576797
472.00 0.082035 1.187069 2.831494 3.867193 6.063035 8.424534
472.50 0.079202 1.165741 2.787914 3.812161 5.961652 8.282925
473.00 0.077538 1.149507 2.740362 3.750914 5.856902 8.143670
473.50 0.076996 1.135963 2.691465 3.689712 5.753216 8.014836
474.00 0.077467 1.124832 2.643794 3.626629 5.658951 7.893250
474.50 0.077394 1.109002 2.601289 3.562375 5.566747 7.781103
475.00 0.078557 1.094735 2.571581 3.503290 5.474199 7.667822
475.50 0.081072 1.081535 2.551920 3.444979 5.382296 7.550396
476.00 0.082832 1.065496 2.531475 3.388122 5.301017 7.422898
476.50 0.083098 1.046970 2.504803 3.335241 5.227389 7.302013
477.00 0.082523 1.027164 2.480674 3.291482 5.155138 7.198735
477.50 0.082591 1.007323 2.455450 3.252002 5.085266 7.099115
478.00 0.081909 0.989143 2.429680 3.215858 5.018255 7.001949
478.50 0.080900 0.973354 2.404356 3.176445 4.958068 6.916981
479.00 0.079080 0.957897 2.379455 3.135794 4.907095 6.839887
479.50 0.076659 0.945676 2.364848 3.093705 4.858003 6.767306
480.00 0.076272 0.935497 2.355429 3.050350 4.803622 6.694711
480.50 0.074426 0.926002 2.345201 3.007163 4.757615 6.612657
481.00 0.072125 0.918301 2.330621 2.963062 4.713423 6.530074
481.50 0.069559 0.911399 2.312179 2.924292 4.664414 6.450526
482.00 0.067773 0.904471 2.289224 2.895079 4.606850 6.373684
482.50 0.065922 0.896640 2.263807 2.872859 4.543268 6.299136
483.00 0.064724 0.886670 2.234906 2.850302 4.473668 6.229722
483.50 0.064889 0.876728 2.200258 2.829106 4.408878 6.158192
484.00 0.063948 0.867439 2.169798 2.805534 4.344413 6.086715
484.50 0.064583 0.854850 2.145287 2.779804 4.277265 6.014202
485.00 0.064009 0.840840 2.121537 2.752895 4.218881 5.937835
485.50 0.064643 0.825958 2.103259 2.721192 4.168304 5.873851
486.00 0.066108 0.811122 2.086251 2.687226 4.128233 5.813181
486.50 0.068161 0.798821 2.070989 2.656340 4.092333 5.757264
487.00 0.070126 0.786967 2.058376 2.629232 4.061081 5.711160
487.50 0.071301 0.775375 2.045972 2.604352 4.032446 5.671963
488.00 0.072878 0.766198 2.029892 2.581274 4.008126 5.632790
488.50 0.072788 0.760072 2.012953 2.555965 3.980830 5.590720
489.00 0.072334 0.755532 1.999528 2.531326 3.954601 5.535734
489.50 0.068607 0.751542 1.981887 2.510179 3.923733 5.468444
490.00 0.066046 0.747299 1.964610 2.489841 3.884455 5.405712
490.50 0.065106 0.742297 1.943104 2.469419 3.843859 5.340091
491.00 0.065232 0.740020 1.924001 2.449360 3.796521 5.271697
491.50 0.065791 0.735078 1.909306 2.431854 3.745246 5.215323
492.00 0.068121 0.727868 1.900010 2.417185 3.697327 5.170391
492.50 0.071863 0.720770 1.892082 2.402916 3.653334 5.132563
493.00 0.075672 0.712789 1.885303 2.379989 3.611121 5.103104
493.50 0.079394 0.704316 1.881637 2.349144 3.584452 5.069952
494.00 0.080268 0.695391 1.874674 2.316365 3.564069 5.022875
494.50 0.079475 0.686384 1.864988 2.283875 3.545234 4.971731
495.00 0.078245 0.679397 1.847599 2.252210 3.532038 4.920075
495.50 0.076277 0.675597 1.826918 2.220414 3.514622 4.869704
496.00 0.072367 0.670359 1.804214 2.194253 3.488290 4.824022
496.50 0.067996 0.664834 1.783339 2.179730 3.457019 4.782542
497.00 0.063936 0.661928 1.763531 2.175062 3.422583 4.748275
497.50 0.061170 0.661056 1.745632 2.173006 3.382648 4.718746
498.00 0.059845 0.661081 1.729174 2.172541 3.352517 4.685534
498.50 0.058976 0.661538 1.712899 2.175151 3.330111 4.637928
499.00 0.058475 0.661417 1.694747 2.180739 3.320443 4.576885
499.50 0.059934 0.663243 1.673112 2.185242 3.325691 4.508523
500.00 0.063000 0.668000 1.648000 2.185000 3.338000 4.437000

Analysis

UV-Vis

Figure 1: Absorbance of Each Lysozyme Standard as a Function of Wavelength of Incident Light (nm)


The figure above shows the absorbance of each lysozyme standard as a function of the wavelength of incident light. The absorbance values were corrected for the water blank and dark noise.


Table 4: Absorbance of Blank (HPLC Water) and Each Lysozyme Standard at a Wavelength of 280nm


The table above shows the absorbance of each lysozyme standard at a wavelength of 280nm. This data is shown graphically in the figure below.


Figure 2: Absorbance of Each Lysozyme Standard as a Function of Concentration (µM) at a Wavelength of 280nm


The figure above shows the absorbance of lysozyme as a function of its concentration at a wavelength of incident light of 280nm. A trend line was added and set to a y-intercept of 0. The equation for this trend line, along with its r squared value, is shown on the graph.

Fluorescence

Figure 3: Fluorescence as a Function of Wavelength for Each Lysozyme Standard


The values for fluorescence were corrected for the water blank.

The area under the fluorescence curve was integrated for each lysozyme standard, giving the intensity of the the fluorescence for each sample. A table and a plot of the intensity as a function of concentration are below.


Table 5: Intensity of Re-emitted Light from Each Lysozyme Sample as a Function of Concentration (M)



Figure 4: Intensity of Re-emitted Light as a Function of Concentration (M)


Matt Hartings Lots of data here! You don't have to use the raw data for your spectra. Just show your graphs. In your A vs conc graph, show your data as points only. Keep the line from the fit. What is your measured molar absorptivity M-1 cm-1?


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