Evaluating Methods of Phycocyanin Extraction and Purification

Introduction: Previous experiments have determined that the difference between certain sonication methods, the ultrasonic bath and the blender which have primarily been used this semester, is potentially unsubstantial. The testing of samples has also been indicating misinformation in the past since it has only recently been realized that 1:10 dilution of samples is necessary to achieve accurate and consistent results. Due to these conclusions, it was tested this week whether the three methods of sonication conducted thus far in the semester display similar changes over time purity values and concentration prior to any other experiments being done. Methods: Art prepared the stock solutions that have been made in the past month, which had been stored at -20 degrees Celsius, by leaving them at room temperature on magnetic plates in order to continuously stir each solution for the next 80 hours. Each container was covered in aluminum foil to prevent light degradation. Three solutions were tested; the solution sonicated with a blender for a total of 30 seconds made on January 28th 2022, the solution sonicated for three minutes with a blender on February 4th 2022, and the solution sonicated with the ultrasonic bath for one hour, made on February 11th 2022. After approximately 80 hours each sample had one milliliter removed and put in an Eppendorf tube to be centrifuged for five minutes at 14,000 RPM at room temperature. Each sample had a duplicate to confirm accuracy. Dilutions of 1:10 ratios were made in order to test on the Nanodrop 2000. After another 48 hours of the same treatment, samples were prepared identically and tested again in order to evaluate any further changes. Results: After 80 hours the February 11 batch had an average purity of 0.416 and the February 4 batch had an average of 0.322 which shows an increase in average purity values, in comparison to a previous range of about 0.37 to 0.39, and 0.28 to 0.32 determined in prior weeks of testing. The increase was more prominent in the February 11 batch rather than the February 4 solution. The January 28 batch had an average purity of 0.259 which was slightly decreased in value compared to the previous 0.280 average. After 128 hours each batch experienced a decrease in purity values except the January 28 batch which stayed nearly identical to the testing two days prior. The February 11 solution decreased significantly to an average of 0.384 while the February 4 batch decreased only slightly to 0.31. 1:10 Dilutions after 80 hours A620 A280 A652 Purity Concentration Purity AVG Concentration AVG 11-Feb 0.278 0.676 0.109 0.411 0.042384644 0.411 0.042384644 11-Feb 0.28 0.665 0.112 0.421 0.042492884 0.421 0.042492884 4-Feb 0.274 0.853 0.126 0.321 0.040126592 0.321 0.040126592 4-Feb 0.278 0.862 0.128 0.323 0.040698127 0.323 0.040698127 28-Jan 0.147 0.572 0.055 0.257 0.022646067 0.257 0.022646067 28-Jan 0.154 0.59 0.061 0.261 0.02342434 0.261 0.02342434 1:10 Dilutions After 128 hours 11-Feb 0.269 0.678 0.109 0.397 0.040699251 0.397 0.040699251 11-Feb 0.261 0.689 0.09 0.379 0.04088764 0.379 0.04088764 4-Feb 0.252 0.811 0.111 0.311 0.037338202 0.311 0.037338202 4-Feb 0.26 0.842 0.119 0.309 0.038126217 0.309 0.038126217 28-Jan 0.148 0.564 0.062 0.262 0.022211985 0.262 0.022211985 28-Jan 0.143 0.559 0.059 0.256 0.021541948 0.256 0.021541948 Conclusion: The results indicate that the amount of time the solution has been sonicated likely alters the purity values that can be attained for that solution. The January 28 batch experienced a decrease from the initial readings a few weeks prior, but virtually no change between 80 and 128 hours during this test. As opposed to the February 4 batch which showed slight difference, and the February 11 batch which showed the greatest difference during this process. This suggests that the method to which the solutions are stored, either temperature, pH, buffer, or any other circumstances can be improved upon. The January 28 batch seems to have hit the low plateau whereas the February 11 batch has the largest rate of decline, with the February 4 batch being a moderate rate between the two. Further testing should be done to determine optimal storage conditions so that purity levels can be maintained and perhaps improved by doing a similar process to this one, which may have promise for increasing purity levels as it did with the February 11 batch initially.