The first thing we can look at in our data is the difference between the DIB spectrophotometric assay and the UV/VIS spectrophotometer. This first thing of note is that between the two, the UV/VIS had an uncertainty that was consistently smaller than that of the DIB spectrophotometric assay. This means that the DIB spectrophotometric assay was less precise than that of the UV/VIS.
This increase in uncertainty could be due to several factors. Looking at the DIB spectrophotometric assay, one of the issues we ran into was when we took a picture of the 96-well plate. When taking this picture, no matter how well the phone was angled, there appeared to be lines and/or multi-colored interference patterns present on the plates. Due to the way that Fiji works, it takes an average intensity over a specific area. With the waves, we are seeing slightly darker or slightly lighter regions which will cause the average intensity to be more biased in one direction. Along with that, these lines were not present in every well, some wells had a perfect blue color and no interference, while the ones next to them could be completely distorted. These differences led to the standard deviation associated with each being much larger than normally would be expected.
Now although the DIB spectrophotometric assay may be less precise, there are some advantages to using it. The biggest advantage is that the DIB spectrophotometric assay is a lot cheaper to run and is a lot more portable. All that is needed is a cheap tablet, a cardboard box, and a camera. For the UV/VIS spectrophotometry, it requires the use of a bulky spectrophotometer that is unable to be transported easily and typically cost thousands of dollars.
Now, when we start to compare the standard addition and external calibration, we are seeing some other differences. One of the first things we see is that the external calibration consistently had higher numbers than that of the standard addition in terms of the amount of aspartame in equal sweetener. Along with that, in the external calibration, there was a greater difference between the two methods used with it. Now what interesting about these differences is that our data may be being affected by the matrix effect.
The matrix effect has the possibility to either enhance the results, in this case increasing the final value for our aspartame concentration or to suppress them. This happens whenever there are other components in the matrix that have the possibility to change the results that we are seeing. One of the biggest issues when using external calibration is that it doesn’t always take this into account. In the external calibration method, we are comparing our sample with the sweetener stock solution with pure aspartame stock solution. In the case of our experiment, we see that by doing this, we get a much higher value for aspartame in our sugar packets.
By running a standard addition calibration, we are attempting to negate these effects. We are doing this by including the sweetener stock solution in the initial sample. Using this, we can compare how our results change by the addition of more aspartame stock solution. When we do this, we see that our value is less than that of the external calibration and most likely, more accurate since it should remove some of the matrix effects.
Now, the last thing of note is that on the packets the company claims that these sugar packets have zero calories. The sugar in these packets is made up of dextrose, maltodextrin, aspartame, acesulfame potassium, and may contain phenylaniline. Of those components, dextrose and maltodextrin are known as carbohydrates. According to sources online, a single packet of equal sugar packets contains about 0.9 grams of carbohydrates. 10 This is equivalent to about 3.6 calories and about 15.1 kJ of energy. This means, that each sample is not truly zero calories.
In the end, we were able to see the advantages and disadvantages associated with each of the various methods we investigated. If we are trying to use the cheapest and most portable set up, our best bet would be to make use of DIB spectrophotometric assay, since it only requires a few different instruments that are cheap to collect. If we want to have the most accurate and precise results, based on what we found, our best bet would end up being to use standard addition with UV/VIS spectrophotometry.