Evaluating Optimal Methods of Phycocyanin Extraction and Purification
Introduction
Previous trials have indicated that in order to optimize phycocyanin extraction from Spirulina plantesis and also achieve desired purity values with consistent repeatability, further focus is required to determine ideal testing methods and crude extract methodology. In an attempt to accommodate for future large scale production, the blending method has been used with high volumes of deionized water, as well as testing of samples done using both the Nanodrop One and Nanodrop 2000. This week adjustments were made to evaluate potential differences in blending times in terms of purity values, concentration values, and overall phycocyanin absorbance (absorbance at 620 nm). Further investigation was also conducted to finalize an opinion for sample testing, as there have been discrepancies between Nanodrop One and the Nanodrop 2000 in previous experiments.
Methods
The initial crude extract was developed by Artemio Chavez by sonicating 40.41 grams of dried spirulina with 400 ml of deionized water. The solution was blended a total of three minutes, in one minute intervals. A ten milliliter sample was aliquoted from the crude solution after each interval, for a total of three samples which will be referred to as one minute, two minute, and three minutes respectively, corresponding to their total sonication times. From these ten milliliter samples, dilutions of 1:10 ratio were made and stored in two milliliter Eppendorf tubes. All six samples were tested on Nanodrop One, and recorded for average purity and concentration values. All tubes were stored at -20 degrees Celsius for three days.
The samples were removed from the freezer and allowed to thaw at room temperature while covered in foil to prevent light degradation. The 1:10 dilution samples were completely thawed and inverted repeatedly for one minute to promote homogenization within the samples prior to testing on the Nanodrop One and Nanodrop 2000. Testing between the two machines were done within thirty minutes of each other in order to ensure accuracy in comparisons. The 1:10 dilutions were then centrifuged for ten minutes at 14,000 RPM at room temperature in an effort to quickly purify that samples. The pellets were identical and a dark blue/green color and relatively small in volume. The supernatant of each sample was removed and tested on Nanodrop One only, after observing fairly consistent results from prior testing.
The one minute, two minute, and three minute tubes of crude extract were thawed completely and inverted repeatedly for one minute to ensure homogenization of the solution before centrifuging at 6500 RPM for 15 minutes at room temperature. Testing was not done prior to centrifuging because visible masses of matter were observed alone the sides of the tube, and the accuracy of the testing would have been compromised. The pellets produced were relatively large in volume, approximately two to three milliliters in each tube. The supernatants were extracted and from those supernatants 1:10 dilutions were made for testing, using 100 microliters of sample and 900 microliters of deionized water, and inverting each eppendorf tube for one minute to ensure a homogenous solution. The results were recorded using the Nanodrop One.
Results
Figure 1 details the initial observations day of sonication, comparing the Nanodrop One and the Nanodrop 2000 as well as crude extract as opposed to 1:10 dilutions in terms of testing accuracy and repeatability. The tubes that were not diluted had a higher variation in results between even the same sample, and tube 3 was recorded as having a negative value for absorbance at 280 nm. The results were much more logical and invariable with the 1:10 dilutions, and the average highest purity was tube 2 (Figure 2) but the highest concentration value resulted from tube 3.
The tests completed after storage below freezing are shown in Figure 3. The average purity and concentration values were nearly identical between the Nanodrop One and Nanodrop 2000, but the Nanodrop One did show more variability between individual readings. The results were consistent with Artemio's results, with tube 2 having the highest purity of 0.298 and tube 3 having the highest concentration at 0.546. Tube one underperformed for each category. After the centrifuge was completed the purity values increased by roughly 0.01 for tubes 2 and 3, but showed almost no difference in tube 1. The concentrations all decreased by a considerable amount. The tubes in which new dilutions were prepared after centrifugation showed an increase of 0.02 in purity value for each tube, as well as concentration values increasing. Still tube 2 showed the highest purity of 0.323 and tube 3 had the highest concentration of 0.599. Figure 4 is the graph generated by the Nanodrop One, which shows high absorbance values at 220-240 nm and moderate absorbances between the range of 300-450 nm, which are nearly level with the 620 nm and 652 nm values. This graph was nearly identical in proportions for each sample tested on the Nanodrop One.
Figure 1- Initial crude extract results taken by Artemio Chavez
Figure 2- Average evaluations of results taken by Artemio Chavez
New protocols require new solutions. Best of luck!
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