Evaluating Methods for Optimal Phycocyanin Extraction and Purification

 Introduction: 

    Attempts to purify crude extracts produced of the phycobiliprotein phycocyanin (C-PC) found in the microalgae Spirulina plantesis have been previously unsuccessful. The test results have shown that charcoal and chitosan adsorption methods for as purification purposes so far have only a negative effect on purity values, opposite to what is expected. Further testing was done in an attempt to differentiate between purification methods more in depth, beginning with primary method of purification, the charcoal adsorption column. Variations that were of interest included the use of a frit, or potentially filter paper, as well as suspending the charcoal in the crude extract as opposed to just adding charcoal to the column. It is crucial for optimal phycocyanin extraction to be able to purify crude extracts in an efficient and productive manner. 

Methods: 

     Two tubes with one gram of spirulina and ten milliliters of deionized water each were sonicated for one hour in an ultrasonic bath. The tubes were centrifuged for fifteen minutes at 6500 RPM, each supernatant removed an deposited into a new tube for a second identical centrifuge. The secondary supernatant was removed and placed in a tube for storage, which was wrapped in foil and kept refrigerated to prevent C-PC degradation. The initial purity and concentration values were measured in a combined tube (1) with a nanodrop. The five milliliters of crude extract was mixed with 0.3 grams of activated charcoal and shaken continuously for fifteen minutes in a foil wrapped tube. The contents were put into a column with a frit, and allowed to completely filter (2B). A sample of two ml of crude extract was also run through a similar column with a frit, but was not shaken with charcoal (2A). The results were measured with a nanodrop. A second method was tested using identical treatment with the charcoal for five ml of crude extract, but was filtered with filter paper rather than a frit in a column (3B). Crude extract that was not exposed to charcoal was also filtered with filter paper to evaluate any obvious differences (3A). Purity values were taken with a nanodrop. After approximately twenty four hours left at room temperatures, each value was retested with a nanodrop to evaluate any changes in purity values. 

Results:

    Figure 1 shows the starting purity value of the crude extract and average absorbances at 620, 280 and 652 nm. Figure 2 is the tubes that were filtered with frit, one with no charcoal (2A) and one with (2B). Figure 3 is the tubes filtered with filter paper, one without charcoal (3A) and one with (3B). Figure 4 is the values after 24 hours of tubes 2A to 3B being left out at room temperature for any observable differences. Figure 5 is the values read approximately five minutes after Figure 4 was taken. Figure one shows a purity value of 1.908, and after charcoal and just filtering the purity values drop by a significant amount in samples 3A and 3B, while tube 2A goes down in purity slightly and 2B increases in purity. Figure 4 shows the next day results in which 3A and 3B are similar to the previous day, but 2A and 2B appear to be above/near analytical grade. Upon further testing just five minutes later, Tubes 2A and 2B are more similar in purity to 2A and 2B. The samples were spun in the centrifuge for a third time and about two centimeters of sediment appeared to be present. 

Crude Extract	A620	A280	A652	Purity(A620/A280)
	2.267	1.188	2.683	1.908

Figure 1

Tube	A620	A280	A652	Purity(A620/A280)
2A	2.507	1.493	2.359	1.679
2B	1.537	0.689	1.999	2.231

Figure 2

Tube	A620	A280	A652	Purity (A620/A280)
3A	1.307	2.737	0.805	0.478
3B	1.298	2.431	0.787	0.534

Figure 3

Tube	A620	A280	A652	Purity (A620/A280)
2A	2.556	0.735	1.353	3.478
2B	2.183	0.451	1.116	4.84
3A	1.290	2.762	0.740	0.467
3B	1.431	2.966	0.794	0.482

Figure 4

Tube	A620	A280	A652	Purity(A620/A280)
2A	1.869	2.645	1.066	0.707
2B	1.782	2.524	1.105	0.706

Figure 5

Conclusion:

    It appears the results are unreliable, and inaccurate to a degree that is not known yet. The sediment present in the tubes that were previously filtered is a source of obvious testing impairment, and future experiments need to be done taking into account that all sediment must be removed before testing a sample to ensure accuracy. It raises to questions in regards to how many times a sample needs to be centrifuged before all sediment is certainly removed, and potentially the centrifugation processes in this and past tests were not sufficient for reliable results. It still remains to be seen how charcoal or chitosan can actually help achieve a higher purity rather than decreasing purity values. Future tests should continue to delve into these methods to ensure that they cannot be found useful.