Chromatography, Part 2
Thin layer chromatography (TLC) can be used to separate many different mixtures. It is very flexible because several different compounds can be separated from each other in one experiment.
Practically speaking, TLC is often used only as an analytical tool rather than as a method of purification. It is used to quickly determine if a mixture is pure, how many compounds it may contain, and what combination of eluent and stationary phase can be used to separate the compounds. However, TLC often works best with a very small amount of material. Isolating useful amounts of compound sometimes requires other kinds of chromatography.
Column chromatography is another kind of liquid chromatography. It works just like TLC. The same stationary phase and the same mobile phase can be used.
Instead of spreading a thin layer of the stationary phase on a plate, the solid is packed into a long, glass column either as a powder or a slurry. Sometimes these columns are several inches wide and a few feet long. A large amount of material can be purified on a chromatography column.
Instead of letting eluent wick up through the stationary phase, the solvent is poured into the top of the column and allowed to run through by gravity. The same factors of adhesion and solution in TLC apply here. If the same solid phase and liquid phase from TLC are used in a column, the compounds will elute through the column in the same order that they elute across a TLC plate.
Sometimes, instead of letting the eluent run through the column via gravity, the eluent can be pushed through more quickly using an inert gas or an air pump. This method is called flash chromatography. There is sometimes a trade-off between quality of separation and the time it takes to run the column, though.
STOP AND THINK
1. Why might there be less separation between two compounds if they move through the column faster?
2. 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. When you get back, you finish the column. You find that you didn't get very good separation between the compounds. What happened?
3. a) Suppose you are working with a chromatography column that can hold about 20 mL of solvent. You know that a sample is usually dissolved and then poured onto the top of the column before eluting with solvent. You dissolve your sample in 20 mL of solvent and proceed with the experiment. You get very poor results. What went wrong?
b) Meanwhile, Alicia, the annoyingly perfect student in the next hood, dissolves her sample in 1 mL of solvent and runs her column. She gets three pure compounds at the end and the instructor immediately gives her an A in the course. What did she do right?
4. TLC shows that the white powder you received in lab contains three compounds. All of them are colourless. You dissolve the sample, load it onto a chromatography column and begin to collect 0.5 mL samples in small test tubes. After twenty test tubes, you get tired and stop the column. How can you use TLC to determine which test tubes have which compounds in them?
Silica and alumina are not the only possible solid phases. Stationary phases can be purchased that have long carbon chains bonded to silica beads. For example, a C18 column contains beads that have 18-carbon chains attached to them. These stationary phases are powders, like silica, and they can be loaded into a column just like silica can.
A C18 column is an example of a "reverse phase" column. Reverse phase columns are often used with more polar solvents such as water, methanol or acetonitrile.
STOP & THINK
5. In a normal column, the stationary phase is more polar than the mobile phase. Is that true in a reverse phase column?
6. In a normal column, three compounds were eluted in the following order: p-dimethylbenzene, p-dimethoxybenzene, then p-methoxyphenol. What might you expect the order of elution would be on a C18 column?
7. You are trying to elute a sample on a C18 column using 20:80 mixture of water:acetontrile, but the compounds are taking too long to come through the column. What should you do?
There are additional methods of chromatography that you might not do in an organic lab. If you do, it will probably be under specific conditions in which your instructor has developed an exact protocol for carrying out the chromatography. These methods are more time-consuming to get working properly. They also use more expensive equipment that may require special training in advanced courses.
Liquid chromatography is often done with more sophisticated equipment. This kind of method is called "high performance liquid chromatography" or HPLC. Rather than packing stationary phase into a glass column, a steel column containing the stationary phase can be purchased. The column can be plumbed into a system that contains a solvent pump to push eluent through the column. After passing through the column, the liquid may go into a UV spectrometer so you can detect when compounds are eluting from the column. The whole apparatus is controlled by a computer. By clicking a button, you can change how quickly the solvent flows. You can easily change the ratio of solvents in the eluent by clicking a button, too.
In addition to a UV spectrometer, other instruments can be used with an HPLC system to get information about compounds being eluted . One of the most important is mass spectrometry (MS). 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.