Evaluating Phycocyanin Extraction and Purification Methods

 Introduction: 

    Previous experiments have so far established that analyzing phycocyanin extraction requires two major components; approximately 1:10 dilutions of sample in appropriate buffer or deionized water, and repeated readings which can be assessed in averages. Values for purity and concentration in the past few weeks have been consistent between each other, although much lower than anticipated. The previous semester produced crude extracts that were supposedly above even food grade purity requirements,  but the authenticity of  that data is now being questioned. This week primarily focuses on comparing the exact sonication practices from the previous semester, which was primarily ultrasonic bath sonification, while also comparing data from samples centrifuged for different durations of time. 

Methods: 

    Arturo prepared the crude extract using 40 grams of dried spirulina and 400 ml of deionized water, with the addition of 40 grams of three mm glass beads. The solution was placed in the ultrasonic bath at 40 khz, for 60 minutes, at room temperature. Then 40 ml of the extract was centrifuged at 10,380 xg, which is the maximum for this lab, using the Beckman Coulter Allegra X22R F0850 rotor. Samples were taken out at 15 minutes, 30 minutes, and 45 minutes, 1:10 dilutions made from these samples, and measured for purity and concentration values on the Nanodrop One. Tubes were stored at -20 degrees Celsius. 

    A few days later I proceeded to thaw and centrifuge both the diluted and undiluted tubes using a microcentrifuge. Centrifugation was accomplished at 14,000 RPM for minutes at room temperature. The pellets were moderate and looks consistent to previous weeks when this procedure was done. Three fifteen ml tubes were also filled with one gram of dried spirulina and ten milliliters of deionized water, in order to have a further comparison identical to trials I had done in the past. These tubes were placed in the ultrasonic bath for 60 minutes at room temperature, and then centrifuged at 6,500 RPM for fifteen minutes. The pellets were very substantial, approximately two to three ml in each tube. The supernatant from each tube was diluted in a 1:10 dilution with deionized water and tested on the Nanodrop One. 

 Results: 

    The results from Art's initial testing was that all the samples had the same purity and concentration values, being 0.38 in purity and 5.9 mg/ml in concentration. 

    The results I acquired, shown in the figure below, gave roughly the same values that Art had found days before. The purity values of each tube ranged from 3.7 to 3.9, and the concentration values from 0.38 to 6.0, although most of the them were in the 4 to 5 range. The tubes that were sonicated that day  ad nearly identical purity values, and matched the same trends found in the rest of the tubes, 

Original 1:10 Diltuions	A620	A280	A652	Purity	Concentration	Purity AVG	Concentration AVG
(1) 15 Min	3.43	9.21	1.9	0.372	0.473670412	0.374	0.4736379526
	3.41	9.08	1.88	0.376	0.4717003745
	3.44	9.2	1.9	0.374	0.4755430712
(2) 15 Min	3.2	8.62	1.76	0.371	0.4430262172	0.371	0.445588015
	3.23	8.72	1.79	0.37	0.4459812734
	3.23	8.71	1.77	0.371	0.4477565543
(1) 30 Min	3.27	8.73	1.77	0.375	0.455247191	0.374	0.4600424469
	3.32	8.89	1.81	0.373	0.4610599251
	3.33	8.9	1.8	0.374	0.4638202247
(2) 30 Min	2.84	7.51	1.54	0.375	0.3951385768	0.377	0.3842022472
	2.7	7.15	1.43	0.378	0.3786853933
	2.71	7.19	1.45	0.377	0.3787827715
(1) 45 Min	6.42	5.97	6.77		0.6013146067	0.384	0.4907752809
	3.85	10.03	2.09	0.384	0.5354569288
	3.74	2.8	4.11		0.3355543071
(2) 45 Min	3.89	10.26	2.09	0.379	0.5429475655	0.377	0.5182459426
	3.64	9.7	1.97	0.375	0.5067827715
	3.64	9.69	1.99	0.376	0.5050074906
New 1:10 Diltuions from Crude Extract
(1) 15 Min	4.26	11.14	2.43	0.382	0.5820561798	0.381	0.5773258427
	4.22	11.14	2.43	0.379	0.5745655431
	4.21	11.03	2.4	0.382	0.5753558052
(2) 15 Min	3.92	10.43	2.23	0.376	0.5361385768	0.38	0.543102372
	3.98	10.45	2.25	0.381	0.5455992509
	4	10.48	2.27	0.382	0.5475692884
(1) 30 Min	2.78	7.29	1.55	0.381	0.3830149813	0.386	0.4004244694
	3.1	7.97	1.7	0.389	0.4296254682
	2.81	7.27	1.55	0.387	0.3886329588
(2) 30 Min	3.98	10.11	2.27	0.394	0.54382397	0.387	0.529835206
	3.83	9.83	2.14	0.389	0.5272734082
	3.74	9.88	2.05	0.379	0.5184082397
(1) 45 MIn	3.58	9.16	2.21	0.391	0.4742434457	0.39	0.4728439451
	3.43	8.79	1.89	0.39	0.4745580524
	3.39	8.7	1.86	0.39	0.4697303371
(2) 45 Min	3.99	10.31	2.24	0.387	0.5483595506	0.391	0.5689550562
	4.32	10.92	2.39	0.396	0.5968426966
	4.08	10.46	2.28	0.39	0.5616629213
New 1:10 Diltuions from 15 ml tubes
1	4.26	11.12	2.37	0.383	0.5873820225	0.384	0.5946741573
	4.36	11.26	2.42	0.387	0.601670412
	4.31	11.24	2.39	0.383	0.5949700375
2	4.37	11.42	2.43	0.383	0.6026554307	0.382	0.6042971286
	4.48	11.66	2.49	0.384	0.617928839
	4.31	11.33	2.42	0.38	0.5923071161
3	3.54	9.3	1.94	0.381	0.4907191011	0.379	0.4917041199
	3.56	9.47	1.96	0.376	0.4926891386
	3.55	9.38	1.95	0.379	0.4917041199

Conclusion:

    From the data it is concluded that the results from last semester cannot be trusted, although the readings had seemed consistent at the time, the dilutions clearly show that there is a discrepancy in accuracy when using undiluted supernatant as the test sample. Though unfortunate, what counteracts that realization is that now we are able to get very consistent values in crude extracts, almost no matter what method of sonication or amount of centrifugation is done. Going forward, methods of extraction can be more accurately evaluated in a DOE, potentially a repeat of previous DOE experiments conducted, so things such as glass beads and phosphate buffer may be permanently reintroduced given new data. It should also be noted that even though the Nanodrop One gave consistent results in terms of averages, the Nanodrop 2000 should likely be used going forward due to random inaccurate readings taking place during this week's testing.