[Kinetics of Crystal Violet Fading]
Roger: Wow, what cool, colored materials do we have here today?
Caroline: I know.
This is purple.
It's my favorite color.
What you're looking at, it's crystal violet.
It's a dye that's used to study tissues under the microscope,
so it's called a histological dye.
What we're going to use crystal violet for today is to study the kinetics of the dye fading.
The bleaching agent that we're going to be using today is sodium hydroxide.
Sodium hydroxide makes the dye fade.
If you ever wanted to do a kinetic study at home and didn't have access to crystal violet,
your cupboard with food coloring will be just fine.
You can study kinetics of food dye fading with a little bit of household bleach.
Works exactly the same way.
Roger: Cool.
Caroline: Let's get started today.
Crystal violet, the first thing that we have to do whenever we do any kind of spectrometer
or colorimeter studies is to figure out what the appropriate wavelength is that the dye
will absorb in.
After all, you don't want to be reading it as the wrong wavelength.
We are using a Pasco colorimeter, a wireless colorimeter, very easy to use.
I will take one sample of our crystal violet, and let's look exactly where the absorbance happens.
We've got some bars.
Let me use the toolbar over there to scale the numbers up.
Roger: Caroline, this looks purple, and I see the biggest bar on there is yellow.
What's that about?
Caroline: That is a very smart question, Roger.
Rather than give you the answer directly, I want you to think about why you would have
yellows in absorbance.
Here's the hint.
Think about the color wheel.
When you look at the color purple, what color is being transmitted and what color is being absorbed?
That should help you answer.
For the purposes of this experiment, now that we know that 570, the yellow wavelength, is
the wavelength that we will be needing to do this study, we now are ready to run our
first experiment for today.
That is to develop a standard curve for crystal violet.
Roger: While Caroline opens up the file that we can run for an exam of different concentrations
and what color they look like, we're going to take these samples that you see in the back.
The darkest one is the most concentrated.
We'll work our way down to the least concentrated so that we understand how actual dark they
are or how much gets absorbed out of that light.
Caroline: I have set up a configuration file to do this study.
I have five concentrations of crystal violet, 15, 18, 20, 23, and 25 micromolar.
You're probably asking the question, "Wait a minute.
We are going to be doing kinetics.
We are not looking for an unknown.
Why are we setting up a Beer's Law calibration curve?"
The answer for that is very simple.
Whenever you do a kinetic study, you have to be very, very conscious of the concentration
of dye.
Why is that, Roger?
Roger: If you're going to have the dye fade, and it's so dark you can't see through it,
you won't know that you've gotten any lighter until you finally get down to the last little bit.
If you want it to be linear, you want to start from the place that the light can see through
it and start fading from there.
Caroline: We want to pick a concentration along the calibration curve that is dark enough
that we will see a change in the color of that dye over time when we add a bleaching agent.
Shall we get started?
Roger: Let's do it.
Caroline: All right.
Why don't you put our lightest sample there, which is 15 micromolar.
Roger: 15 micromolar.
Caroline: This is the absorbance of 15 micromolar.
Let's try the next one up.
This is the absorbance of 18 micromolar.
Let's try the next one.
Roger: You can see the dots showing up on the right side there of the graph.
OK, here's
the 20 micromolar.
Caroline: 20.
Roger: There's the 21.
Caroline: 23.
Roger: 23 micromolar.
The final 25-micromolar solution.
Caroline: Perfect.
We're going to stop this right now.
We're going to look at our dots, and we are going to use one of the tools to see how linear
that curve is.
Now we're going to select one of these concentrations to use for our kinetics.
In this case, we are using the highest concentration, which is 25 micromolar.
Let me explain to you what we did with this.
For the second part of this experiment, we're going to be doing kinetics.
In this part, we have pipetted, into each of our cuvettes, exactly one-and-a-half milliliters
of your 25-micromolar solution of crystal violet.
We are going to be adding to this an equal molar of sodium hydroxide.
The only difference is the concentration of sodium hydroxide.
Let me tell you how this is all set up.
Every cuvette is going to have one-and-a-half milliliters of 25-micromolar crystal violet,
and it's going to be diluted by half.
You're going to have to calculate what your final crystal violet concentration is.
The solutions of sodium hydroxide that will be added to this are 0.02, 0.1, 0.2, and just
for kicks, I brought a little bit of cheap drain cleaner from the house.
We're going to check all of them and see what kinetics look like.
Remember the concentrations of sodium hydroxide that we gave you.
If you notice, you will also be diluting them by half in each of these runs.
When you do your analysis, make sure that you are aware of what that final concentration
of sodium hydroxide is.
Shall we get started?
Roger: Let's get started.
Caroline: Our first one, we will add one-and-a-half...By the way, let me show you what we did with this.
I tried to do this lab by counting dots, but I realized that I was so distractible that
I used to lose count of the dots.
What I did was I took pipettes and I simply calibrated them to give me exactly one-and-a-half mils.
Roger is now going to pipette.
Before we do that, I think we need to set up and look for our other graph.
Roger: This I want to pull in the amount up to the red line of the sodium hydroxide.
I'll put this into the solution, and that will slowly cause that purple crystal violet
to fade.
We're going to put this in the cuvette reader, and we're going to watch as that yellow absorbance
starts to come through.
Caroline: All right.
Roger: Remember, yellow's being absorbed, and it's going to go away.
Caroline: Are we ready?
Roger: I think so.
Caroline: OK.
We're going to work as swiftly as we can.
This is why it's great to have a partner like Roger with you.
I'm going to put that in, and we're going to press the start button.
Roger: To give you a feeling while that's going, I'm going to do this also in the video camera.
You can watch to see how long that takes to also lose its coloring.
You can see that process happening in the camera as well as watching it happening on
the graph.
It's going down a little bit.
This is also the least concentrated because we're using an eyedropper that I can't clean
between times for the film's purposes, so we're going to go to more concentrated, more
concentrated, which should leave the least amount of error as I go up concentration.
Caroline: We're going to let this go about 100 seconds.
Roger: This may take two or three of our experiments to see finally it change.
We've put the least concentrated sodium hydroxide into that one.
I think it's getting less visible when we mix the next one.
Caroline: We're getting a slope there.
Roger: Yeah, there we go.
While that runs those last few seconds, I'll get the sodium hydroxide ready for the next run.
Here's going to be more concentrated.
Caroline: We're going to stop right there.
Let's pull that puppy out.
We can leave it laying down here.
Let's see what happens to it over time.
Now he's going to add one-and-a-half milliliters of 0.1 molar sodium hydroxide.
Roger: This is more concentrated.
I'll let that stir just a little.
It appears to be getting lighter.
Caroline: Mm-hmm.
I hope the yellow is showing very well.
Roger: Solutions are purple again because the yellow is being absorbed, so we're following
that yellow absorbance that's getting less and less yellow absorbed from there.
We'll put it next to it on this napkin here in a second.
While that's finishing up, I'll get the last more-concentrated sodium hydroxide solution ready.
Then we can follow this and see how that rate changes by putting in more-concentrated sodium hydroxide.
Caroline: As you guys can tell, there is a noticeable rate change between the first rung
and the second rung.
Push that right there.
Now we're getting ready to add the highest concentration of sodium hydroxide that we have.
Roger: What's nice about being able to measure these is that you can see changes.
If you look at the ones that are on our napkin, it may be difficult to quantify how different
those are, which makes little instruments like these a great help in studying these
types of reactions.
While that's finishing up, I have one last solution, and that's drain cleaner.
That's a little more concentrated yet.
Caroline: Correction, cheap drain cleaner.
Roger: Treap chain drain.
Caroline: Cheap drain cleaner.
Roger: That's right.
What do you think is going to happen as I add this more concentrated solution to my
crystal violet?
You'll have a minute to think about that while we finish this last reaction out.
Caroline: This is a good time to predict whether you think drain cleaner has a higher sodium
hydroxide concentration than 0.2.
We're going to stop right there.
Let's take this puppy out, lay it down.
Whoa, look how much lighter it is now.
Roger: Yeah.
Caroline: Here's the old drain cleaner, or the inexpensive drain cleaner.
Cap this right here.
While this is running, I want to throw out some questions.
You are AP, you are advanced students, and this is a very good time to starting thinking
about how you want to analyze this data.
First of all, as you look at the curves coming about, do you notice if they are absolutely
linear, or are they non-linear?
If they are non-linear, it is really important for you to select a portion of the curve that
shows you the greatest change in rate.
This is going to be the initial rate in your situation.
Whenever you study rate data, you've got to make sure that you are picking the portion
of the curve where you're getting the maximal change in Y for the X that you're going through.
Roger: Visually, as she has mentioned, the rate change is getting clearer faster or the steepest.
You can see as we got more concentrated.
This was the least-concentrated solution.
This is the medium-concentrated solution.
This is the most concentrated solution.
You can see visually what that's ended up doing as I'm sitting here and while this is
running and you're seeing happening on a graph.
Caroline: One of the challenges of studying kinetics is to be able to predict what the
order of your reaction is.
Your challenge is to take this data and convert the Y-axis from absorbance to natural log
of absorbance and also to convert it to one over the absorbance.
The linearity of your curves when you do that kind of analysis is going to determine whether
or not your reaction's a zero-order, a first-order, or a second-order reaction.
For now, let us look at what all of our curves look like,
There's a huge change in rates.
Your job is to now do analysis on this and look at what the reaction order is.