Evaluating Methods for Optimal C-PC Extraction

Introduction: 

In previous weeks, there have been varied results between multiple methods of phycocyanin extraction from dried Spirulina plantesis. Last week, it was decided to begin a three factor DOE experiment in order to determine which factors are the most significant in the extraction process. The three factors in question were the presence of glass beads as opposed to none, the use of a blender in addition to the ultrasonic bath versus only utilizing the ultrasonic bath, and phosphate buffer rather than deionized water. These factors, A, B, and C, were tested in eight runs and two trials. 

Methods:

Tubes 5A through 8B were centrifuged at 9500 RPM for 30 minutes at 4 degrees Celsius and their supernatant extracted and put into new tubes. All of the tubes were then centrifuged at 6500 RPM for fifteen minutes, to complete their second round of centrifugation. The supernatant was extracted from each tube and tested for absorbance values at 620 nm, 280 nm, and 652 nm using a nanodrop. These values determined the purity (A620/A280) and concentration mg/ml ((A620 - .474(A652))/5.34). 

Results:

The data shows that the only statistically significant factor in terms of purity was the use of glass beads. The statistical effect of glass beads was approximately 0.42, nearly double the value of the decision limits determined. Neither phosphate buffer or blending was shown to be significant, with effects of -0.004 and 0.0415 respectively. Their combinations were also insignificant with combination AB -0.132, AC -.00275, BC, -0.214, and ABC 0.00675. It was noted that the highest purity achieved was tube 7B, which had the phosphate buffer, glass beads, and the ultrasonic bath alone, but 7A was treated identically and was much more similar to the other tubes. The lowest value recorded was from tube 4A, which was treated with water, no beads, and the ultrasonic bath alone. In terms of concentration, none of the factors tested were shown to be statistically significant. Factor A had the effect of -0.09875, B had 0.2335, C 0.17875, AB 0.072, AC 0.22875, BC -0.0435, and ABC -0.2265. It was noted that the tube with the lowest purity, 4A,  also had the highest concentration of 3.366 mg/ml. The tube with the lowest concentration value was 3B, treated with water, beads, and ultrasonic bath at 2.157 mg/ml. 

 

A620

A280

A652

Purity

Concentration mg/ml

1A

2.568

1.402

2.478

1.832

2.609

1B

2.747

1.374

2.410

1.999

3.005

2A

2.429

1.610

2.336

1.509

2.475

2B

2.717

1.552

2.130

1.751

3.197

3A

2.512

1.369

2.702

1.835

2.306

3B

2.342

1.264

2.511

1.853

2.157

4A

2.603

2.289

1.700

1.137

3.366

4B

2.323

1.629

2.254

1.426

2.349

5A

2.869

1.522

2.201

1.885

3.419

5B

2.267

1.409

2.000

1.609

2.470

6A

2.576

1.664

2.070

1.548

2.987

6B

2.460

1.810

1.865

1.359

2.951

7A

2.733

1.458

2.423

1.874

2.967

7B

2.841

1.232

2.782

2.306

2.851

8A

2.508

1.906

2.509

1.316

2.469

8B

2.624

1.477

2.404

1.777

2.780


The data below exhibits the statistical significance of factor A (beads), factor B (blender), and factor C (phosphate buffer) on the purity levels of the tubes tested. 


The data below shows the statistical significance of factor A (beads), factor B (blender), and factor C (phosphate buffer) on the concentration levels of the tubes tested. 


Discussion: 

    The results have resolved some of the issues presented by this experiment, in that the difference between phosphate buffer and water is incredibly minimal, as well as the use of a blender rather than the ultrasonic bath. The 50 ml volume was potentially a limiting factor for the blender such that a higher volume of solution may have been more effectively blended, since 50 ml was hardly even touched by the blades of the blender. It is also suggested to utilizing the blending and ultrasonic bath method whenever possible because it reduces the total sonicating time by nearly half, if not more, and can potentially eradicate the need for glass beads, if the volume is great enough. In smaller volumes, of 100 ml or less, glass beads and the ultrasonic bath alone are suggested. The use of phosphate buffer can also be deemed unnecessary, due to the fact that deionized water can nearly replicate buffer’s results. The results of this experiment determined there may be a difference in preferable methods depending on volume, which should be tested further. Ideally, there would also be more trials run to further ensure the accuracy of results. 

Comments

  1. EricaNovember 20, 2021 at 3:54 PM

    Very nice work Leilani. Love all the data and stats. It will be interesting to see what happens as we scale up!

    ReplyDelete

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