Thin-layer chromatography (TLC)
Thin-layer chromatography (TLC) is a technique used to separate mixtures of compounds based on differences in polarity. In TLC, a glass plate coated with a stationary phase (typically silica gel) is spotted with the mixture to be separated. The plate is then placed in a mobile phase (solvent), which travels up the plate by capillary action. The rate at which each compound in the mixture moves up the plate depends on its relative attractions to the stationary and mobile phases. Created by Sal Khan.
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- After watching the videos, my understanding is that TLC doesn't actually identify the precise substances in a compound. Instead it demonstrates the relative properties of a compound, and the number of constituent elements within the compound.
Is this correct?
Or is there a standard metric that you can use to identify which element is in the compound based on the Rf value?(3 votes)
- So on it's own chromatography(doesn't matter the specific type) all achieve the same goal of separation of compounds in a mixture. So if you just separated the compounds, then yes that wouldn't really help you determine the identity of the compounds. Maybe just their polarity relative to each other.
Actually identifying the compound results from the retention factor we calculate. Each compound has a characteristic retention factor which is constant (assuming you use the same mobile and stationary phase). So if you know the retention time of a compound from chromatography you have a good chance of identifying it.
Hope that helps.(5 votes)
- Isn't this kind of like gel electrophoresis?(3 votes)
- Gel-electrophoresis and thin-layer chromatography, TLC, are similar in that they are both separation techniques. They are able to separate the components of a mixture for analysis.
Gel-electrophoresis separates large macromolecules, usually DNA fragments and proteins, based on their molecular mass. A mixture is placed into the gel wells and a current separates them because the macromolecules are charged (like the negatively charged phosphate groups in DNA) so they migrate either towards the positive or negative terminal. Molecules with smaller molecular masses migrate farther since they are lighter while the heavier molecules remain close to the injection point. This separates the molecules into bands where we can determine approximately how massive each of the bands are.
TLC separates molecules in mixtures based on their polarities. The plate itself is coated in a material which together constitutes the stationary phase. This plate is placed into a solution which constitute the mobile phase. The stationary and mobile phases are constructed of materials with different polarities; so one will be nonpolar and the other will be polar. Before the two phases come into contact, the bottom of the stationary phase is spotted with drops of the mixture. The mobile phase climbs up the stationary phase using capillary action and carries with it the spots. Certain components are more attracted to the mobile phase than to the stationary phase and travel high up the plate with the mobile phase. The components which do not travel far are more attracted to the stationary phase and the mobile phase has a comparatively smaller pull on it. Depending on the polarities of the two phases, we can determine how polar each of the components are.
So, being separation techniques both gel-electrophoresis and TLC separates mixtures into their constituents. Gel-electrophoresis uses electricity and separates based on molecular mass, while TLC uses capillary action and separates based on polarity.
Hope that helps.(3 votes)
- Why is the stationary phase traditionally a polar substance and the mobile phase traditionally a nonpolar substance? Can the polarities of the phases be reversed (i.e. a nonpolar stationary phase and a polar mobile phase)?(3 votes)
- For chromatography like TLC, there are two main types; normal-phase and reverse-phase.
In normal-phase the stationary phase is polar and the mobile phase is less polar than it (mind you the mobile phase does not have to be nonpolar, just less polar than the stationary phase). The most commonly used stationary phase in normal-phase is silica gel like what was used in the video here. The most common solvent mixtures include ethyl acetate/Hexanes for less polar compounds and methanol/dichloromethane for more polar compounds.
In reverse-phase the stationary phase is nonpolar and the mobile phase is more polar than it. One of the more common stationary phase is C-18, which is essentially a nonpolar hydrocarbon. The most common solvent mixtures are water, tetrahydrofuran (THC), acetonitrile, or methanol.
Hope that helps.(1 vote)
- um sorry can i ask why the mobile phase actually moves the substance(s) upward at all?(1 vote)
- The mobile phase moves up the stationary phase via capillary action. As it moves, the chemicals being analyzed feel a force of attraction to the mobile phase because of intermolecular forces. So essentially the analyzed chemicals are dragged up the plate by the mobile phase because of those attractive forces.
Hope that helps.(3 votes)
- is that correct that the more soluble a substance is, the less it's polar(1 vote)
- The solubility of a chemical depends on the solvent. Depending on the solvent, being less polar can help or hurt a chemical's solubility. The general rule being "like dissolves like", or polar solvent dissolve polar solutes while nonpolar solvents dissolve nonpolar solutes.
Hope that helps.(1 vote)
- [Instructor] So let's say that I have a vial of some mystery liquid right over here, and I want to start figuring out what's going on there. And the first step is to think about, is it just one substance or is it a mixture of multiple substances? And the focus of this video is a technique to separate out the substances to understand at least how many there are, and this technique generally is called chromatography, but we'll focus on thin layer chromatography which is the most common that you might see, but other variations of chromatography like paper chromatography operate on very similar principles. So what we're going to do is set up on top of something like glass or plastic, we're going to put a thin layer of a solid polar substance. Now, what you typically do is put a thin layer of silica gel, that's the most common solid polar substance that folks use. And it's also porous. And the fact that it's porous is really important because we're going to want liquid to have capillary action and travel up through it. Now, the silica gel, as I mentioned, this thing is very polar. Now, what we're going to do is take some of our mystery substance, let's say it's this color right over here, and we're going to place a dot of it on that silica gel. You then want to take this plate that has the silica gel on it and that little dot of our mystery substance, and then you want to dip just one end of it in a solution. And what's really important is that the solution is less polar than the silica gel. Less polar here. And we'll talk a little bit about what happens depending on how polar this is. Now, usually this is going to be a very shallow amount of this solution, which, as we'll see, will be something of a solvent. And you usually want to put it in a closed container like this so that this fluid down here doesn't evaporate out. And then what do you think is going to happen? Well, as I mentioned, this is a porous substance here. And so you're going to have capillary action. This fluid at the bottom is going to move upwards through the silica gel, through those little pores in the silica gel. This is the stationary phase. Why do we call it that? Well, 'cause it's not moving. And you can imagine we would call this less polar solvent the mobile phase, because that is traveling through the silica gel and it's picking up some of this mystery substance and it's transporting it. And let's say this mystery substance is made up of two different things. If something is more polar, that means it's going to be more attracted to the stationary phase which is very polar. And so it's not going to travel that far, while the parts of our mystery substance that are less polar, they're not going to be attracted to the silica gel as much. So they're going to travel further with the solvent. So maybe it might go like that. And you would run this until your mobile phase makes a good way to the top of your silica gel right over here. Now, just looking at this, and the reason why it was called chromatography is when they originally did this, they were actually separating out various tissues in vegetation that had different colors. The chroma is referring to the various colors, but it doesn't necessarily even have to refer to things that have different colors or sometimes you might need a UV light to see them. But when you run thin layer chromatography, you will see that your original dot will have traveled to various degrees with your solvent and then will now be multiple dots depending on how many things were in your original mixture. And as I just mentioned, this thing right over here, this is the less polar thing is going to travel further than the more polar thing, more polar constituent substance, because the more polar thing is more attractive to the silica gel, which is stationary, and there is a way to quantify how far these things traveled relative to your solvent. And that's called a retention factor. Retention factor. Which the shorthand is R subscript f. And it's just defined as the distance traveled by the solute divided by the distance traveled by the solvent. And we need to be clear. It's not the distance traveled by the solvent in total, it's the distance traveled by the solvent from this origin, from where we applied this dot right over here. So, past the origin. And let me label that as the origin. So what would it be in this situation? Well, to help us there, we would have to get out a ruler. So the retention factor for substance A right over here, so I'll put that dot there, label that A, would be equal to the distance traveled by the solute, which we can see, it traveled one centimeter, one centimeter, over the distance traveled by the solvent past the origin. And so that is going to be, we see it traveled five centimeters past the origin. So one centimeter over five centimeters, which is the same thing as 0.2. And then the retention factor for substance B is going to be equal to, how far did it travel? Well, it traveled three centimeters out of a total of five centimeters for the solvent, past this origin, past where we put the sample right over there. Five centimeters, which is equal to 0.6. So notice, in this situation, the more polar substance had a lower retention factor than the less polar substance, and that makes sense. Because our stationary phase is more polar than our solvent, and so the things that are more polar were harder to move by the less polar solvent.