Calculation Results

Alright, so down to some number-crunching!

The magic formula I used for calculating percent starch was:

% Starch = [(∆Atest )(900) ]/  [(∆Astd)(sample weight in mg)]

(I won't bore you with details on where the 900 came from but it involves the multiplication of molecular weight of starch monomers, initial sample volumes from glucose sample volume assays, dilution factors, and whatnot.)

Okay. The values for the 1 mg sample, 5 mg sample, and 10 mg sample turned out to be 24.3%, 33.7%, and 56.7%.

Plotted on a graph, the relationship turns out to be somewhat linear, which is a good and expected:

Next week I will repeat the standardization experiment to make sure everything is correct (and to make sure it is repeatable), before moving on to the real samples.

Pink is My Favorite Color (Day 2 Standardization Continued)

Check out these beauts!

Fig. 1 (From left to right): Standard Blank, Standard, Reagent Blank, Cornstarch Samples "1", "5", "10"

The intensity of the pink color pictured above is proportional to the original glucose percentage. I got absorption readings of 0.02, 1.2, .185, .22, .41, and .95, respectively (at 540nm).

Also, this was my work area:

Fig. 2

...Cozy, no?

(That labeled bottle on the left is the sulfuric acid solution! And the paper on the right - that's my procedure bible.)

I'm going to take these measurements home and plug them into my calculations, and see what I'll get for the percent starch. 

This is so cool!!! :)

Day 2 of Starch Assay Standardization

So today I was back in 201P finishing up the remainder of last night's procedure. The first thing I did was turn on the Spectrophotometer (Spec 20) and let it warm up for 15 minutes. Then I retrieved my samples and pipetted the solutions - my Standard Blank (distilled water), Glucose Standard (water + the glucose that came in the kit), Reagent Blank (the blank from the Starch digestion - contains only the reagents), and the 3 cornstarch samples - into their respective cuvettes.

This step was all about timing. If we let the reaction for each sample go on for differing amounts of time, we aren't going to be evaluating them on the same standard. So upon adding the Glucose Assay Reagent to each sample, I had to incubate each tube for exactly 30 minutes in a 37˚C water bath; then, I had to stop each reaction by adding 2ml of the 12N Sulfuric Acid solution.

I figured the easiest, most efficient way I could do this was by adding the Glucose Assay Reagent to the first tube (standard blank) mix, and then put it in the incubator; wait 1 minute, before adding the reagent to each subsequent tube. Then, when time was up for the first tube, I would add the H2SO4, take that out, and then wait another minute; that's how I knew the 30 minutes was up for the second tube, and so on and so forth.

But I'm not going to lie - it was pretty nervewracking. I don't think my eyes ever left the screen of my i-phone timer.

An Elaboration

I thought I'd take a moment to clarify what this procedure is all about.

Essentially this starch assay is a reaction in 3 steps: 

I stopped right after the first reaction; after the fourth reaction, I will be left with samples that will be pink in color, in varying shades. I will be using a spectrophotometer to measure the absorbency of the samples, and this measurement will be proportional to how much starch is in each. 

IT'S GETTING REAL

My ordered starch assay kit came in the mail yesterday!
And so, today I stayed after school from 3 until 6pm to perform my first ever starch assay, but first on a control (cornstarch) to standardize my procedure. I used 1 mg, 5 mg, and 10 mg samples of cornstarch for this experiment.

Today basically involved a lot of precise massing, measuring, tube-cleaning, labeling, incubating, timing, diluting, pipetting, adding, mixing, waiting, and pipetting some more, transferring...and a lot of reconstituting materials beforehand - all the reagents, the 80% ethanol solution, and the 12N Sulfuric Acid solution... (<-- Yikes I know)

(Actually, after Dr. B finished making the 12 N H2SO4 solution, I felt the bottle, and it was hot. Like, hot hot. Hot like a stove. Or a steaming mug of hot chocolate. Or your favorite actor/actress! Or my school's theater when the heater goes on overdrive! ...You get the idea. )
(And that is why, folks, always remember to add acid to water, and not the other way around. #lessonsfromAPchem)

It was a 3 part procedure (one that I had typed up and tailored a month prior). The first part was the sample preparation; this time around it was extremely manageable as I was already working with finely ground cornstarch. The second part, was the starch digestion. Order matters here: I wound up adding first 80% Ethanol solution, then alpha-amylase (then heating the enzymatic reaction), then adding what was titled, "Starch Assay Reagent" (then incubating that), and finally diluting the samples and proceeding to the glucose determination, part 3.

ACTUALLY IT IS IMPORTANT TO NOTE that I stopped right after finishing Part 2 today, due to time constraints (Basically, it was 6pm and if I stayed any longer, the school would have probably kicked me out.)

So, after diluting each sample with distilled water, the last step of Part 2, I refrigerated the samples overnight at around 2-8˚C - not exactly the original plan, but hopefully it won't impact my results too much. Fingers crossed, and we'll see how tomorrow morning goes...

Good News and Bad News...

So after returning back from my wonderful, feast-filled, stomach-stuffing and much-needed Thanksgiving break, I came back to Prep Room 201P... and guess what  surprise was waiting for me:

Fig 1: 15s flask with unidentified cloudy substance

A STRANGE YELLOWISH-BROWNISH FOREIGN CONTAMINANT!!! Those pesky bacteria!

Or maybe it was a fungus... I'm not sure.

But, in any case, the good news was that the duckweed colony was proliferating!!! ;D

Originally only one frond was placed into the flask - now, there were 5. Go asexual reproduction, go!

The other samples also showed signs of growth:

Clockwise from upper left: 30s, 1m, 1m30s

For now, I am just going to let the samples grow and not worry about the mysterious intruder in yellow.