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# Spectrophotometry and the Beer–Lambert Law

Spectrophotometry is a technique that uses light absorption to measure the concentration of an analyte in solution. The amount of light absorbed by a solution is related to the analyte concentration by the Beer–Lambert law, which is expressed as follows:

*A*=*εbc*, where*ε*is the molar absorptivity of the analyte,*b*is the path length (the distance the light travels through the solution), and*c*is the concentration of the analyte. Created by Sal Khan.## Want to join the conversation?

- About Absorbance at7:57. When Sal says "The negative log of T" does he mean you take the logT and multiply it by negative 1 (-1), or does it mean 1 over the log of T (1/logT)? Like how 10^-1=1/10.(4 votes)
- He means either (-1)×logT or log(1/T).

Note: log(1/T) = log1 - log T = 0 - log T = -logT.

They both mean the same thing.(18 votes)

- Hi Sal, in my A-level course we have to learn the colors of transition metals and about d-d transition and metal-ligand charge transfer, is there any way to be able to calculate the colors/wavelengths? as its hard to remember the color changes as my mind is quite mathematical(15 votes)
- On an atomic level, are bonds non-transparent too? And can you concentrate a liquid to where no light can get through?(4 votes)
- Electromagnetic bonds are indeed invisible. It's like holding two magnets a couple inches apart; there's nothing holding them together but the electromagnetic force. You probably could concentrate a liquid so no light got through: if the solution is 99.99 solvent and the rest solute, then you essentially have a solid block which no light can get through.(7 votes)

- On the graph at11:01, is there a unit in which absorption is measured?(4 votes)
- According to

https://in.answers.yahoo.com/question/index?qid=20061018151521AAIBvPP

Absorbance doesn't have any units because its the ratio of the amount of light that passes through a solution compared to the amount of light that is passed into it. Sometimes you'll see absorbance units (AU) as its units.

Hope this helps :)(2 votes)

- at5:19he uses the word opaque?? what does that mean?? less solutable??(2 votes)
- Hi Zack,

An opaque object does not allow light to pass through it (ie. a deck of cards, a sheet of aluminum foil, etc.). An easy way to check for opacity is to completely cover a lit flashlight with the object in question. If no light passes through, the object is opaque. However, if some light, or all the light, can pass directly through the material, the object is not opaque.

Opacity can be compared to transparency and translucence. A transparent ("see-through") object allows all light to pass through it (ie. a sheet of plastic or clear glass), and a translucent object allows only some of the light to pass through it (ie. a sheet of wax paper).

Here's a good article with some more on opacity, including a quantitative definition: http://en.wikipedia.org/wiki/Opacity_(optics)

Hope that helps :)(3 votes)

- At1:28something was said about shining a wavelength of light is specifically sensitive to the solute. my question is why is the spectrophotemeter set at certain wavelengths, why cant it just be set at any wavelength of our choice?(2 votes)
- That is because chemicals only absorb very specific wavelengths of light. This is why we can use a spectrophotometer to measure the concentration of a specific chemical. If the wavelength of the light is wrong, then the light won't be absorbed.(3 votes)

- thanks for your answer but just one more thing. What i asked abt the frequency ,is it true in the case of wavelength(2 votes)
- Frequency and wavelength are just different ways of expressing the same thing. Frequency = c / wavelength (where c=speed of light).(2 votes)

- What is molarity? Sal didn't mention it any of the previous videos, so I'm surprised at it showing up so suddenly. Can anyone help?(2 votes)
- Molarity is a measure of concentration of a solution and is measured by moles of a solute per liter of solution. so a 2.0M NaCl solution (say "two-point-zero molar") contains two moles of NaCl for each 1 liter of solution.

For more information, look for videos on solutions.(2 votes)

- what unit is light measured in and with what instrument ?(1 vote)
- I probably sound immature but what do those little shapes that look like fish at9:30mean?(1 vote)
- It's a sign that means proportional, as in A is proportional to l*c(2 votes)

## Video transcript

- [Instructor] What I want
to do in this video is to talk a little bit
about spectrophotometry. Spectrophotometry, photometry, which sounds fairly sophisticated, but it's really based on
a fairly simple principle. So if I have, let's say
we have two solutions that contain some type of solute. So that is solution one, and
then this is solution two. And let's just assume that our
beakers have the same width. Now let's say solution,
let me put it right here. Number one, and number two. Now let's say that solution one has less of the solute in it. So let me, let me make... So that's the water line right there. So this guy has less of it,
and let's say it's yellow, or to our eyes, it looks yellow. So this has less of it. So this hasm actuallym
let me do it this way. Let me shade it in like this. So it has less of it. And let's say solution number
two has more of the solute, so it's more. So I'll just kind of represent that as more closely packed lines. So the concentration of
the solute is higher here. So let me write higher concentration. Higher concentration. Concentration, and let's say, and this is a lower, lower concentration. Now let's think about what will happen if we shine some light
through each of these beakers, and let's just assume that we
are shining at a wavelength of light that is specific to the... That that is specifically
sensitive to the solute that we have dissolved in here. But I'll just leave that
pretty general right now. So let's say I have some
light here of some intensity. So let's just call that... Let's call that the incident intensity. I'll just say that it's
I0, so it's some intensity. What's going to happen as the light exits the other side of this beaker right here? Well, some of it is going to be absorbed by our little molecules inside the beaker, so you're going to have less light come out to the other side. I'll call this I1. Now, in this situation, if
we shined the same amount of light into this beaker, so
it's the same number that is, that is the same, the
same intensity of light, what's going to happen? Well, more is going to
be absorbed as the light travels through this beaker. It's just going to bump
into more molecules, 'cause it's a higher concentration here. So the light that comes out when you have a higher concentration, I'll
call that the intensity, I'll call that I2, this is going to have a lower intensity of light
that's being transmitted than this one over here. In this case, I2 is going
to have a lower intensity, is going to be less than I1. If you have another beaker
that is maybe twice as wide, it's twice as wide, and
let's say it has the same concentration as number
two, we'll call this one number three, it has the same
concentration as number two. So I'll try to make it
look fairly similar, and you were to shine some light in here. Let's say you shine
the same light in here, and you have some light that
makes it through, that exits, and then this is actually
what your eyes would see. So this is I3 right there. What do you think's going to happen? Well, it's the same
concentration, but this light has to travel a further
distance of that concentration. So once again, it's going
to bump into more molecules and more of it will be absorbed, and so less light will be transmitted. So I2 is less than I1, and I3, I3 is actually going to be the least. And if you were looking at
these, this has the least light, this has a little bit more
light being transmitted, this has the most light being transmitted. So if you were to look at this, if you placed your eyeball right here, this one right here would
have the lightest color. You're getting the most
light into your eye. This would be a slightly darker color, and this would be the darkest color. That makes complete sense. If you dissolve something,
if you dissolve a little bit of something in water, it will
still be pretty transparent. If you dissolve a lot
of something in water, it'll be more opaque. And if the cup that you're dissolving in, or the beaker that you're
in, gets even longer, it'll get even more opaque. So hopefully, that gives you the intuition behind spectrophotometry. And so the next question is, well, what is it even good for? Why would I even care? Well, you could actually
use this information. You could see how much
light is transmitted versus how much you put
in to actually figure out the concentration of a solution. That's why we're even talking about it in a chemistry context. So before we do that, and I'll show you an example
of that in the next video, let me just define some,
really, some terms of ways of measuring how concentrated this is, or ways of measuring how
much light is transmitted versus how much was put in. So the first thing I will
define is transmittance. And so when the people
who defined it said, well, you know, what we care about
is how much is transmitted versus how much went in, so
let's just define transmittance as that ratio. So in this example, the
transmittance of number one would be the amount that got through over the amount that you put in. Over here, the transmittance
would be the amount that you got out over the
amount that you put in. And as we see, this one right
here will be a lower number. I2 is lower than I1, so this will have a lower
transmittance then number one. So let's call this transmittance two. This is transmittance one. And transmittance three is
the light that comes out, that gets through, over
the light that goes in, and this is the smallest
number, followed by that, followed by that. So this will have the least transmittance, it's the most opaque, followed
by that, followed by that. Now another definition,
which is really kind of a derivative of the transmittance, and not in the calculus sense, it's just derived from
transmittance, and we'll see, it has pretty neat properties,
is the notion of absorbance. And so here, we're trying to measure, how good is it at absorbing? This is measuring, how good
are you at transmitting? A higher number says
you're transmitting a lot, but absorbance is how
good you're absorbing, so it's kind of the opposite. If you're good at transmitting, that means you're bad at absorbing, You don't have a lot to absorb. If you're good at absorbing, that means you're not transmitting much. So absorbance. Absorbance, right here,
and absorbance is defined as the negative log of transmittance. And this logarithm is base 10, or you could view that if the
transmittance, we've already defined as the negative, the
negative log of the light that is transmitted over
the light that is input, but the easiest way is the negative log of the transmittance. And so, if transmittance
is a large number, absorbance is a small
number, which makes sense. Now, what's also cool about this. is there something called
the Beer-Lambert law, which you could verify, and this is... We'll actually use this in the next video, Beer-Lambert law. I actually don't know the
history of where it came from, and I'm sure it's based
on somebody named Beer, but I always imagined it's
based on someone transmitting light through beer, the Beer-Lambert law. And this tells us, this
tells us that the absorbance is proportional to the path length. So this would be, how far
does the light have to go through the solution? So it's proportional to the path length times the concentration,
times the concentration. Usually we use molarity
for the concentration. Or another way to say it
is that the absorbance is equal to some constant. It's usually a lowercase
Epsilon like that. Some constant, and this is
dependent on the solution, or the solute in question,
what we actually have in here and the temperature and the
pressure and all of that. It's equal to constant times
the length it has to travel, times the concentration. Let me make it clear right here. This thing right here, this thing right here is concentration. Concentration. The reason why this is super
useful, as you can imagine, so let's say we have an axis right here. That's axis, and over here,
I'm measuring concentration. This is our concentration axis, and we're measuring it as molarity. And let's say the molarity starts at zero. It goes, you know, I
don't know, .1, .2, .3, so on and so forth. And over here, you are
measuring absorbance. In the vertical axis
you measure absorbance. Now, let's say you have some solution, and you know the concentration, you know it is a .1 molar concentration. So let me write down M for molar. And you measure its absorbance and you just get some number here. So you measure its absorbance
and you get its absorbance, so this is a low concentration,
didn't absorb that much. You get, I don't know some number here, so let's say it's .25. And then let's say that
you then take another known concentration, let's say .2 molar, and you say that, oh
look, it has an absorbance right here at .5, and I should
put a zero in front of these. 0.5 and 0.25. What this tells you, this
is a linear relationship. For any concentration, the absorbance is going to be on a line. And if you want a little
review of algebra, Epsilon times the length
will be the slope. But the important thing to realize is that you have a line here. You have a line here, and why? And the reason that's useful
is you could use a little bit of algebra, figure out
the equation of a line, or you could just look at it
graphically and say, okay, I had two known concentrations, and I was able to figure
out the absorbance. You can then go the other way around. You could then measure for
some unknown concentration. You could figure out its absorbance. So let's say there's some
unknown concentration, and you figure out its
absorbance is right over here. Let's say it's .4. Then you can just go on this
line right here, and you say, okay, well then that must be, that must be a
concentration of this, well, whatever number this is. And you could measure it, or you could actually
figure it out algebraically. And so this will be
pretty close to .2 molar, a little bit less than 0.2 molar. And we're gonna actually
do an example of that in the next video.