>> Hello and welcome to the Penguin Prof Channel.
This is part of my laboratory success series.
And we're going to talk about how you use a standard curve
to find the concentration of materials in an unknown sample.
We're going to do it Penguin Prof style, of course.
And, if you would, take a second and show your support
by clicking those buttons.
I would really appreciate it.
Thanks. You want to stay tuned to answer these questions.
How do you find out how much stuff you have in your sample?
What is a standard curve?
How do you use the standard curve?
And we're going to look at, as an example,
finding the concentration of protein in an unknown sample.
So how you measure anything, basically,
you're taking a series of things whose values you know.
And comparing them to the thing with the value
that you want to know.
I know that sounds strange, but it is very confusing
for students just what the standard curve really is
used for.
So I want to compare it to the measurement of length.
And, just because people have a lot more experience with that.
So length is a great example.
If you want to know how long something is,
you obviously, use a ruler.
But have you really thought about what a ruler is?
A ruler is an object with standards of length.
And you compare this set of standards
with the object whose length you want to know.
Now again, I realize this is kind of obvious.
But the question then becomes what kind
of ruler should you use?
Now, if I want to measure the length
of my little penguin earrings, I'm going to use a ruler that is
of appropriate length; right?
I know this is obvious.
But I can't use my little handy-dandy key chain ruler
to measure, for example, the length of a car.
Even this highly penguinized car.
It's just not appropriate.
So you have to have a rule.
And the rule is that the unknown must be within range
of the measuring device you are using.
So this really cool ruler is great for measuring a pen.
But not so great for measuring a car.
And I know this is obvious with length.
But, when you see how standard curves are used,
you'll be amazed how easy it is to violate this rule.
So moving away from length now.
If you want to know the concentration of whatever
in your unknown sample, this is how you do it.
So you have a blood sample.
Or a urine sample.
Or a mucous sample from a patient
or a penguin or whatever.
And we're going to be using a spectrophotometer
to measure the concentration of material in that sample.
This video is not about that, but I will put a link
in the info bar below on a document that I have
that explains the principles
of spectrophotometers and their use.
But basically what's happening is this.
We're using light at a very particular wavelength
to measure absorbance through a sample.
All right, so again, for details on this go ahead
and check out that document.
So here are the steps for making your standard curve
and finding the concentration of an unknown.
You're going to make the standard solutions.
Then you're going to measure the absorbance of those solutions
in the spectrophotometer.
Then you're going to make a graph
of concentration versus absorbance.
That is called the standard curve.
Then you're going to measure the absorbance
of the unknown that you have.
And then you're going to graph it
and determine the concentration.
So we're going to look at all five steps in an example here.
So I make my standard solutions with different concentrations
of the material that I am interested in.
So I am adding the material to each of,
in this case I just picked five, cuvettes.
So I know what's in each one; right?
Because I made them.
I'm going to take each of those vials, and I'm going
to measure them in the spectrophotometer.
And I'm going to graph absorbance.
So now I have a table of concentration
and absorbance for my proteins.
Now all I have to do is graph them.
And you can do this by eye if you're just doing, you know,
a real quick version of this.
Or you can enter it into a computer,
and the computer will tell you the confidence of the line.
And how well the line, you know, fits your data.
But this is basically what you need.
The standard curve is actually not a curve.
You need to use the portion of the curve that is linear.
That is a whole other discussion.
But the standard curve is actually a line, okay.
And, if your data don't make a line, you can't use it, okay.
Now this is the standard curve.
This is your ruler.
So this is the set of standards of known concentration.
In other words, you know what the concentration
in each tube is.
And you know what the absorbance is.
And so that's your ruler.
So now, when you have your unknown, this is step four.
You take your unknown and you put it in the spectrophotometer,
and you get the reading.
And now you put that reading on the Y-axis.
So here I just happened to get 0.70.
Now I put that on the Y. I draw straight out a line
until it hits my standard curve.
And then I draw straight down from there
until it hits the X-axis.
And that value on the X is the concentration of my unknown.
Now, I just need to remind you again of that rule.
That the unknown must fall within the range
of the measuring device.
I know it seems obvious with a ruler,
not so obvious with standard curves.
So let's say that my unknown, instead of 0.70, gave me 1.57?
Now, I have students who do this all the time, okay.
So they just go ahead and extend the Y-axis up like this,
so they can put that absorbance on there.
And then they draw straight across.
And they find, I've got to extrapolate
that standard curve out.
And I'm going to just draw, I mean,
does this look legal to you?
No. You can't do that, okay.
So that's what I mean when I say that what it is
that you are measuring must fall within the range
of the units of measure.
So all you would do in this case is make some more standards;
right?
Standards of higher concentration
so that your unknown does fall on the standard curve.
That's the solution to that problem.
The solution is not extrapolating that line.
And that's it.
As always, I hope that was helpful.
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