>> 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.
Thank you so much for visiting
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