Spectrophotometry

in this video, we're gonna talk about Spectra Fatima tree. So, as you guys probably recall from your previous courses, the light absorb its of a molecule can determine the amount of that molecule, and so proteins can actually be quantified by measuring their light. Absorb INTs and biochemists use specific instruments that air called spec trophy Tom Attar's. In order to acquire these light, absorb INTs values. And then biochemist can use the light absorbent values to determine the concentration of the absorbing salute or the concentration of the protein and so down below. You can see that the relationship between the light absorb INTs and the absorbing saw you concentration is expressed by the Lambert Beer Law, which is also commonly known as just Beer's law. And so down below, in our example image on the right hand side notice we're giving you guys beer's law and also notice that it has all of these different variables that Aaron it. So first, what we're gonna do up above is defined each of these variables as we refresh our memories on how a typical spectra fought ometer works. And then afterwards I'll let you guys know how you should expect to use beer's law moving forward in this course. And so we know that Spectra Fatah motors are used to acquire the light absorbing values. And so the first variable here in beer's law, the A is really just referring to the absorb INTs of the salute or the absorb mints of the protein. And so, with a typical specter of atomic er, there's going to be a light source and down below in our image. This white light is coming from the light source, and the white light can be directed towards a de fracture, which is this triangular prism over here, and the D fracture acts as a wavelength selector. So when the white light is directed towards the D fracture, it will def fracked the light into all of its wavelengths. And then one particular wavelength can be selected to move forward in the process and before this wavelength of light that is selected actually hits the protein sample. It's referred to as the incident light, and the incident light can be abbreviated with the symbol I sub not zero, where zero here means initial or incident. The beginning. And so, looking at Beer's law noticed that we have this eyes of not zero and beer's law and up above, we can indicate that the ice of non zero is really just referring to the initial intensity of light or the incident light. Now notice that here we have our protein sample. And when this incident light actually hits the protein sample that some of our proteins are going to absorb some of that light. But not all of the light is absorbed. Some of the light passes through the sample. Some of the light is transmitted through the sample and notice that this transmitted light here is less intense. Then the incident light. And that's because some of the light was actually absorbed by the protein sample. And again, this light that passes through this transmitted light is actually referred to as the transmitted light. And it could be abbreviated with the symbol I. And so in our beers law equation, notice that we also have this I variable here and so up above, we can indicate that the I variable is really just the transmitted intensity of light. After the incident, light passes through the sample, and so the transmitted light can hit a detector, and the detector can measure the amount of transmitted light. Now, notice again that inside of this container here we have our protein sample. And so what do you guys think? What factors can actually influence the amount of light that's absorbed by the protein sample? Well, one of the factors is actually the concentration of the protein. And so here we have that the concentration of the protein sample is important is an important factor for determining how much light is absorbed and the concentration can be abbreviated with, Ah, see. And so, looking at our beer's law equation over here, notice that we actually have a C over here, because the concentration is going to determine how much light is absorbed. And so the mawr proteins there are the greater the concentration of protein, the cloudier this substance is going to be, the sample would be, and the mawr light it's going to absorb. And so the concentration is definitely a variable that we want to take into account and so up above weaken indicate that sea is the concentration of the absorbing salute. Now, what's another factor that could potentially influence the amount of absorbing, uh, the amount of light that's absorbed by the sample. Well, it turns out that actually the container, the length of this container eyes going to determine how much light is actually absorbed. And so the length of the container is going to dictate the length of the path light the length of the light path. So the longer this container is, the longer the path is that the light has to take to get through the sample. And so literally this distance of the container is a factor, and, uh, the amount of light that's absorbed. The longer that light path is, the mawr light will be absorbed. The shorter that light path is, the less light will be absorbed. And so this length here is a factor, and so weaken abbreviated with the symbol L, and it's typically measured in units of centimeters. And so, looking at our beer's law on the right, notice that we have this cursive L over here, and that's referring to the length of the light path, and again it's typically measured in units of centimeters. Now our last variable here is our trickiest variable because it's not as easily represented in an image down below. And so this last variable is the Greek symbol Epsilon, and it's really just referring to the extinction coefficient. And the extinction coefficient is also known as the absorptive ity of a molecule or of a salute. Now the extinction coefficient is really just a specific property off a salute, and so it determines how much light is actually absorbed, and it's very specific to each molecule. Now we're going to talk a lot more about the extinction coefficient in our next lesson video. But for now, all I want you guys to know is that Epsilon, this Greek symbol here represents the extinction coefficient, and that's a property that controls the amount of light that's absorbed by assault. And so, for that reason, we have to take it into account and the beer's law equation. And so you can see that the Epsilon here is the molar extinction coefficient. Now what? I want you guys to realize about beers laws that there's these two equal signs that air in there, and these two equal signs essentially break our beer's law into three different parts. We have the absorb. It's over here. On the far left, we have the log of the, uh, incident light over the transmitted light in the middle. And then we have the extinction coefficient times, the concentration times the length of the light path on the far right and so moving forward in our practice problems. Depending on the information that's provided in the practice problem, we may not need to use all of these portions of beer's law. Uh, if we're not given any information about the incident light or the transmitted light, then it's likely we're not going to need this portion of beer's law. And we can focus specifically on this portion over here and the absorb. It's over here now, uh, in a similar way. If we're not given the extinction coefficient, then we can assume that we're not going to use this portion of beer's law and that we're probably going to need to use the incident light over the transmitted light. And so, depending on the information that's provided in the practice problem, that's going to dictate which portions of beer's law that we're going to need to use. So, uh, this here concludes our lesson on Spectra Fatima Tree and Beer's Law and and our next lesson video. We'll talk Maura about the extinction coefficient, but before we get there, let's get a little bit of practice, so I'll see you guys in that video.

So now that we've refreshed our memories on Spectra Fatima Tree and we've covered beer's law and this video, we're going to focus on the extinction coefficient, which we know is also sometimes called the absorptive ity and is symbolized with Greek symbol Epsilon. So the extinction coefficient is really just a specific property of a chemical that measures how strongly that chemical absorbs light at a particular wavelength of light. And so the wavelength of light can be symbolized with Greek symbol Lambda shown here. And the greater the Mueller extinction coefficient is, the greater the absorb INTs is going to be. And so the molar extinction coefficient will actually very with different wavelengths of light. And so what this means is that the wavelength of light is going to be very critical to pay attention to moving forward as we use beer's law and the molar extinction coefficient actually has units of inverse morality and inverse centimeters. And so that is also important to take note of, because in our practice problems, using beer's law, we're going to need to make sure that all of the units appropriately matched, and if not, we need to do some unit conversion. Now down below, In our, uh, image, we have a graph being shown. And on the Y axis, we have the extinction coefficient and units of inverse similarity and inverse centimeters. And on the X axis, we have the the wavelength and units of nanometers and which will? Notices were measuring the extinction coefficient of a specific molecules at multiple different wavelengths and notice that the wavelength actually changes the Mueller extinction coefficient. And so again, because the extinction coefficient is involved in beer's law, the extinction coefficient has an effect on the absorb its and so because the extinction coefficient varies with different wavelengths of light, that means that the absorb INTs also varies with different wavelengths of light. And so because the wavelength here is so critical to the extinction coefficient and to the observance, that means that we're gonna need to pay attention closely, toe what wavelength of light were using, and and our next lesson video, we're going to talk about exactly what wavelength of light is typically used toe analyze proteins. And so before we actually get to that lesson video, we'll get a little bit of practice with these concepts here in our next video. So I'll see you guys there

So in our last lesson video, we reviewed the Lambert Beer Law and Spectrum Optometry and Spectrum Optometry data can be plotted onto an absorbent spectrum, and so an absorbent spectrum plots the absorb its values on the Y axis and the wavelength of light on the X axis. And so, looking at our example down below at the absorbent spectrum on the Left, notice that we have the absorb its values on the Y axis and the wavelength of light on the X axis and also noticed that we have two different types of molecules that are being plotted. We have a protein molecule being plotted with the blue curve, and we have a nucleic acid molecule being plotted with the red curve and notice that these two different molecules absorb light strongly at different wavelengths of light. And so the nucleic acid molecule and red absorbs light strongly at a wavelength at about 260 nanometers, whereas the protein molecule strongly absorbs wavelengths of light at 280 nanometers. And it turns out that a peak light absorbent, specifically at 280 nanometers eyes a characteristic property of most proteins and so It turns out that the reason that most proteins strongly absorbed wavelengths of light at 280 nanometers is due to just two amino acids, and those two amino acids are trip to fan and tire scene. And so trip to fan strongly absorbs light at wavelengths of 200 nanometers, 280 nanometers, and tyrosine weekly absorbs wavelengths of light at 280 nanometers. But nevertheless they both absorbed wavelengths of light at 280 nanometers, which is actually a unique characteristic property of these two amino acids. And so we can clearly see that down below in this absorbing spectrum on the right, where again we have the absorbent on the Y axis and the wavelength of light on the X axis, and we can see that trip to fan is strongly absorbing wavelengths of light at 280 nanometers, and tyrosine is weekly, absorbing wavelengths of light at 280 nanometers. Now we know that trip to fan and tire seen are just two of a total of 20 amino acids. So what about all the other amino acids? Well, it turns out that those amino acids do not strongly absorb any characteristic wavelength of light. And so biochemist don't use their light absorbent to quantify the concentration of proteins. But because again trip to fan and tire seen strongly absorbed this unique characteristic wavelength at 280 nanometers, they can use their light absorbent to quantify proteins, and it turns out that most proteins and nature are going to be quite large, and they're gonna have a lot of amino acids. And so the chances that they're gonna have trip to fan entire scene is quite high. And that's why biochemists are able to use wavelength at 280 nanometers to acquire the absorb its of most proteins and to use that absorb INTs to dictate or to determine the concentration of most proteins. But of course, if the protein does not have any trip to fans or any tire scenes in them, then using a wavelength of 280 nanometers is not gonna be the best way to quantify the protein, and they're gonna need to use a different method to do that. And so this concludes our lesson here on why trip to fan and tire seen absorb light strongly at 280 nanometers, and we'll be able to get some practice in our next video, so I'll see you guys there.