Liquid chromatography-mass spectrometry LC-MS can be used to determine the molecular weights of the compounds as they elute. That infromation can be used to help identify the compound. Gas chromatography is an important variation that you should know about. Instead of passing a liquid over the stationary phase, an inert gas moves over the stationary phase.
The inert gas may be helium or nitrogen. The equilibrium here is between compounds absorbed onto the stationary phase and compounds moving in the gas phase. Intermolecular attractions with the stationary phase play a role in GC, but so does the boiling point of the compounds. Because most compounds are not very volatile, they would spend all their time sitting on the solid phase under normal conditions. For that reason, the column in a gas chromatograph is placed inside an oven.
The temperature in this oven is carefully controlled so that compounds will spend a greater fraction of time in the gas phase. The eluent can't be varied in GC. It is just an inert gas. To control separation of compounds in GC, we can change the pressure of the inert gas, which controls how quickly the gas flows. We can also control the temperature, which influences how much time compounds spend moving along in the gas phase. We can also choose different kinds of columns with different stationary phases.
Chris P Schaller, Ph. Suppose you are in the middle of separating a mixture on a chromatography column when you remember that you left the oven on in your apartment. Your apartment is a half hour from lab. You turn off the stopcock at the bottom of the column so that the eluent stops flowing.
In thin layer chromatography, the stationary phase is a thin layer of silica gel or alumina on a glass, metal or plastic plate. Column chromatography works on a much larger scale by packing the same materials into a vertical glass column. Various sizes of chromatography columns are used, and if you follow a link at the bottom of the page to the Organic Chemistry section of the Colorado University site, you will find photographs of various columns.
In a school lab, it is often convenient to use an ordinary burette as a chromatography column. Suppose you wanted to separate a mixture of two coloured compounds - one yellow, one blue.
The mixture looks green. You would make a concentrated solution of the mixture preferably in the solvent used in the column. First you open the tap to allow the solvent already in the column to drain so that it is level with the top of the packing material, and then add the solution carefully to the top of the column. Then you open the tap again so that the coloured mixture is all absorbed into the top of the packing material, so that it might look like this:.
Next you add fresh solvent to the top of the column, trying to disturb the packing material as little as possible. Then you open the tap so that the solvent can flow down through the column, collecting it in a beaker or flask at the bottom. As the solvent runs through, you keep adding fresh solvent to the top so that the column never dries out.
Note: These diagrams are very simplified in order to make them easier to draw. In reality, the colours won't separate out into these neat blocks, but will probably be much more spread out - more so the further down the column they get. This assumes that you have read the explanation for what happens during thin layer chromatography.
A poorly packed column can lead to uneven flow and band broadening, both of which give rise to poor separation. For this method, add your dry solid phase to the column and pass equilibria buffer or starting solvent to saturate the solid. Regardless of how you fill the column, always make sure that there are no bubbles and that the stationary bed is even.
The mobile phase, or eluent, is a solvent or buffer that dissolves your sample and transports it through the column. The eluent can be a pure solvent, a mixture of different solvents, or a buffer that varies in pH and contains additives. Different column chromatography methods call for different mobile phase conditions, so select the type of eluent after you decide on the type of column. Run an isocratic elution for noncomplex samples. The separation will depend on the properties of the molecules and the extent of their interactions with the stationary phase.
Put simply, an analyte that strongly interacts with the stationary phase is retained in the column and, therefore, moves slowly. But when the interactions are weak, the analyte elutes easily and, thus, exits first see Figure 1 below.
I always find it safer to collect fractions from the start of the chromatography column run—better safe than sorry I guess. After that, collect smaller fractions when you begin eluting your material. So let's redraw your silica line in here.
This is now where your silica is, but as you see, the original green band has separated into two distinct bands now. You can kind of see a yellow one and a blue one. In real life, the colors might not be quite this distinct, but you get the idea. And as this proceeds, again you'll see the silica line, but the separation between the two bands will actually become more and more distinct.
And so far what you've been collecting in your flask is mostly all just solvent, but how do we actually collect whatever's in the blue band? And you might not know exactly what compound it is, but you can tell that by the fact that they're traveling at different rates, the blue and yellow bands probably have different polarities and are different compounds. So when you see that the blue band is getting really close to the bottom, you'll want to quickly switch out your old flask for a new one so that you've collected in the new flask, it's just this layer of the blue compound, while in your column you still have the yellow layer.
Note that each flask you collect is considered a fraction, and that's how you conduct column chromatography. So earlier I was telling you want to make sure that these are pretty much horizontal, but what happens if instead when you're packing your column or pouring something in, it ends up looking kind of crooked?
So in this case, let's say that our column was pretty crooked, what you would see instead, this is the silica line. But as those two bands traveled through, instead of seeing them just parallel to one another, and again perfectly perpendicular to your column, what you'd see is something that looks more like this.
You have the yellow band kind of slanted, then you'd also have the green band slanted if you had loaded in your compound in the slanted manner. And the issue with this is that at a certain time point, say if you're trying to collect the fraction that falls between here and here, you're not really getting the pure yellow compound or the pure green compound. Instead, you're getting a mixture of the two, which shows that this isn't a very efficient purification. So what you'll want to do next time is be very careful in the initial stages, because a lot of the work with column chromatography is making sure that you prep it just right.
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