Spectrophotometry - Video Tutorials & Practice Problems
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1
concept
Spectrophotometry
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Video transcript
In this video, we're going to talk about spectrophotometry. So as you guys probably recall from your previous courses, the light absorbance of a molecule can determine the amount of that molecule. And so proteins can actually be quantified by measuring their light absorbance. And biochemists use specific instruments that are called spectrophotometers in order to acquire these light absorbance values, And then biochemists can use the light absorbance values to determine the concentration of the absorbing solute or the concentration of the protein. And so down below, you can see that the relationship between the light absorbance and the absorbing solute 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 are in it. So first, what we're gonna do up above is define each of these variables as we refresh our memories on how a typical spectrophotometer 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 spectrophotometers 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 absorbance of the solute, or the absorbance of the protein. And so with a typical spectrophotometer, 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 diffractor, which is this triangular prism over here. And the diffractor acts as a wavelength selector. So when the white light is directed towards the diffractor, it will the 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 0, where the 0 here means initial or incident, the beginning. And so looking at Beer's Law, notice that we have this I sub not zero in Beer's Law. And up above, we can indicate that the I sub not 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 than 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 can be abbreviated with the symbol I. And so, in our Beer's 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 a c. And so looking at our Beer's law equation over here, notice that we actually have a c over here because the concentration more, proteins there are, the the greater the concentration of protein, the cloudier this, substance is going to be the sample would be, and the more light it's going to absorb. And And so, the concentration is definitely a variable that we want to take into account, and so up above, we can indicate that c is the concentration of the absorbing solute. Now, what's another factor that could potentially influence the amount of absorbing, the amount of light that's absorbed by the sample? Well, it turns out that actually the container, the length of this container, is 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 in, the amount of light that's absorbed. The longer that light path is, the more 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 we can, abbreviate it 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 absorptivity of a molecule or of a solute. Now, the extinction coefficient extinction coefficient is really just a specific property of a solute, and so it determines how much light is actually absorbed, and it's very specific to each molecule. Now, we're gonna 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 a solute. And so for that reason, we have to take it into account in 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 Beer's Law is that there's these two equal signs that are in there, and these two equal signs essentially break our Beer's law up into 3 different parts. We have the absorbance over here on the far left. We have the ins the log of the, 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. If we're not given any information about the incident light or the transmitted light, then it's likely we're not gonna need this portion of Beer's Law. And we can focus specifically on this portion over here and the absorbance over here. Now, Beer's law, and that we're probably going to need to use 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, this here concludes our lesson on spectrophotometry and Beer's law, and in our next lesson video, we'll talk more 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.
2
Problem
Problem
What is the relationship between light absorbance (A) & the amount of light transmitted through a sample?
A
Increased transmitted light results in increased A.
B
Decreased transmitted light results in decreased A.
C
Decreased transmitted light results in increased A.
D
Transmitted light & A should always remain equal.
3
concept
Spectrophotometry
Video duration:
2m
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Video transcript
So now that we've refreshed our memories on spectrophotometry and we've covered Beer's law, in this video, we're gonna focus on the extinction coefficient, which we know is also sometimes called the absorptivity and is symbolized with the 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 the Greek symbol, lambda shown here. And the greater the molar extinction coefficient is, the greater the absorbance is going to be. And so the molar extinction coefficient will actually vary 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 molarity 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 match. And if not, we need to do some unit conversion. Now down below in our image, we have a graph being shown, and on the y axis we have the extinction coefficient and units of inverse molarity inverse centimeters, and on the x axis we have the, the wavelength in units of nanometers. And what you'll notice is we're measuring the extinction coefficient of a specific molecule at multiple different wavelengths. And notice that the wavelength actually, changes the molar extinction coefficient. And so, again, because the extinction coefficient is involved in Beer's law, the extinction coefficient has an effect on the absorbance. And so, because the extinction coefficient varies with different wavelengths of light, that means that the absorbance also varies with different wavelengths of light. And so, because the wavelength here is so critical to the extinction coefficient and to the absorbance, that means that we're gonna need to pay attention closely to what wavelength of light we're using. And in our next lesson video, we're gonna talk about exactly what wavelength of light is typically used to 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.
4
Problem
Problem
Which of the following options is false for Beer's Law?
A
Absorbance increases as concentration increases.
B
Absorbance decreases as path length increases.
C
Absorptivity is wavelength specific.
D
Absorbance spectrums plot absorbance and wavelength.
5
concept
Spectrophotometry
Video duration:
4m
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Video transcript
So in our last lesson video, we reviewed the Lambert Beer law and spectrophotometry. And spectrophotometry data can be plotted onto an absorbance spectrum. And so an absorbance spectrum plots the absorbance values on the y axis and the wavelength of light on the x axis. And so looking at our example down below at the absorbance spectrum on the left, notice that we have the absorbance values on the y axis and the wavelength of light on the x axis. And all also notice that we have 2 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 2 different molecules absorb light strongly at different wavelengths of light. And so the nucleic acid molecule in 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 absorbance, specifically at 280 nanometers, is a characteristic property proteins strongly absorb wavelengths of light at 280 nanometers is due to just 2 amino acids, and those 2 amino acids are tryptophan and tyrosine. And so tryptophan strongly absorbs light at wavelengths of 200 nanometers 280 nanometers, and tyrosine weakly absorbs wavelengths of light at 200 80 nanometers. But nevertheless, they both absorb wavelengths of light at 280 nanometers, which is actually a unique characteristic property of these 2 amino acids. And so we can clearly see that down below in this absorbent spectrum spectrum on the right, where again, we have the absorbance on the y axis and the wavelength of light on the x axis, and we can see that tryptophan is strongly tyrosine is weakly absorbing wavelengths of light at 280 nanometers. Nanometers. Now, we know that tryptophan and tyrosine are just 2 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 biochemists don't use their, light absorbance to quantify the, concentration of proteins. But because, again, tryptophan and tyrosine strongly absorb this unique characteristic wavelength at 280 nanometers, they can use their light absorbance to quantify proteins. And it turns out that most proteins in 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 tryptophan and tyrosine is quite high, and that's why, biochemists are able to use a wavelength at 280 nanometers to acquire the absorbance of most proteins and to use that absorbance to, dictate or or to determine the concentration of most proteins. But, of course, if the protein does not have any tryptophans or any tyrosines in them, then using a wavelength of 200 80 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, tryptophan and tyrosine 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.
6
Problem
Problem
A) Suppose myoglobin's molecular weight is 17,800 g/mole and its extinction coefficient at 280 nm wavelength is 15,000 M-1 cm-1. What is the absorbance of a myoglobin solution (concentration = 1 mg/mL) across a 1-cm path?
Hint: Use Beer's law.
a. 0.49
b. 0.73
c. 0.36
d. 0.84
B) What is the percentage of the incident light that is transmitted through this solution?
a. 14%
b. 6%
c. 21%
d. 58%
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7
Problem
Problem
A protein solution has an absorbance of 0.1 at 280 nm with a path length of 1 cm. If the protein sequence includes 3 Trp residues but no other aromatic residues, what is the concentration of the protein? (Trp ε = 3,400 M-1 cm-1).
A
29.4 μM
B
5.32 μM
C
9.8 μM
D
0.7036 μM
8
Problem
Problem
An unknown protein has been isolated in your laboratory and determined to have 172 amino acids but does not have tryptophan. You have been asked to determine the possible tyrosine content of this protein. You know from your study of this lesson that there is a relatively easy way to do this. You prepare a pure 50 μM solution of the protein, and you place it in a sample cell with a 1-cm path length, and you measure the absorbance of this sample at 280 nm in a UV-visible spectrophotometer. The absorbance of the solution is 0.398. How many tyrosine residues are there in this protein? (Tyr ε ≈ 1,000 M-1 cm-1 ).