Ryan Lo

Ryan Lo
Canadian International School of Hong Kong

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Questions (3)
Question
I'm currently looking at the rheological properties of the polymer Xanthan Gum. focusing on its dynamic viscosity to be more specific. I'm assessing the effects of pH (ranging from 3.6 to 5.6, 0.4 increment, total of 6 pH's) on the dynamic viscosity of xanthan gum solution (dissolving xanthan gum powder into acetic buffer with equal ionic strength, concentration is kept at 0.04%).
Firstly, my viscosity data collected shows that, as pH increases from 3.6 to 4.0 then 4.4, the viscosity increases; but as I bring up the pH from 4.4 to 4.8, 4.8 to 5.2, then lastly 5.2 to 5.6, the increasing viscosity trend plateaus and the increase in viscosity is less significant compared to the 3.6-4.4 jump. At this range, does pH has an effect on the viscosity of xanthan gum based on its molecular configuration? Though some sources states that xanthan gum's viscosity remains stable and unchanged within the range of pH 3-12 at a high concentration like 1% not 0.04%, yet some suggest pH still plays an effect, though I'm not sure how on the chemical and molecular aspect.
A possible conjecture I can think of is the xanthan gum's order-disorder and helix-coil transition is affected by protonation. In figure 2, it demonstrates how electrolytes affect the structure of the polymer; in figure 3, it shows how at a state of a helical rod and no longer a random coil, it is capable to hydrogen bonds among each other. Hence, I'm wondering of pH plays an effect on it's structural transition, such that the increased intermolecular forces at the form of a helical rod would make it more viscous in solution.
Here are the resources I have used so far:
Brunchi, CE., Bercea, M., Morariu, S. et al. Some properties of xanthan gum in aqueous solutions: effect of temperature and pH. J Polym Res 23, 123 (2016). https://doi.org/10.1007/s10965-016-1015-4
Question
I'm currently using Bradford method for a protein assay. And I am measuring the proteolytic activity in roots of certain plants, as many studies found plants have in situ proteases to aid the nitrogen uptake. To determine the proteolytic activity, I will incubate the roots in a known ovalbumin standard solution. Then, after a period of time, the Bradford Assay will be conducted on the incubated solution to look for a decrease in the concentration. I am stilldetermining what concentration to use. In case you don't know, the dye itself without proteins has a maximum absorbance at 495nm, with proteins, it will have a maximum absorbance at 595nm. The dye also has a protein detecting range of 200 - 1500 μg/mL, which is the case for albumins (often BSA is used, but I was restricted to use ovalbumin). When I tested the ovalbumin solution with a concentration of 1500 μg/mL, no color change could be seen, the peak from spectrophotometric analysis also remained at 495nm. I then tested with a range of ovalbumin solutions, then realising with a concentration at or above 50mg/mL, a color change can be seen. The expected results are, there should be a color change & a peak at 495nm when the ovalbumin concentration is at 1500 μg/mL, but there wasn't, and it also requires a high concentration to detect protein concentration, which is odd for a sensitive assay like the Bradford method. Do you have any insights or ideas of what is causing this problem? My current idea is, there is something abnormal about the dye (this is the product I'm using from Bio-rad), or the ovalbumin powder that I used to make the solution has impurities. The impurities idea might be incorrect though, as the powder I'm using is lab grade, and the impurities shouldn't be too significant to this extent.
Question
I am currently conducting a protein assay with the Bio-Rad Protein Assay Dye Reagent Concentrate, it's active ingredient is Coomassie Brilliant blue G-250. When distilled water is added to the concentrated dye according to the ratio given on the Bio-Rad manual (1 part of concentrated dye 4 parts of dH2O), the colour changed from its darkish-red to a lighter brown colour. Then, to test how the water affects the dye, I continued adding more distilled water. As more water is added, the colour changed from light brown to green, then from green to aqua, then aqua to blue. When the colour stopped changing, no more water is added. The whole volume of the water-dye solution is now around 50mL, and it appears to be light blue. Before & during & after adding distilled water, the water-dye solution has remained at a pH of 1.5 (due to the presence of phosphoric acid in the dye). Is this colour change normal when water is added? This may or may not be related to the of the expiration by 1.5 years from its 1 year shelf life. Thanks.