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. Keep in mind that collecting more and smaller fractions may give you more chances of isolating your material free of contaminants. 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. Once you know this, you can combine all of the samples which contain your pure product, and then remove the solvent. How you would separate the solvent from the product isn't directly relevant to this topic and would vary depending on their exact nature - so I'm not even going to attempt a generalisation.
Note: If you don't know how do make a thin layer chromatogram , you should have followed the link I gave you earlier! Anyway, here it is again. You will find detailed descriptions with photographs of how to carry out column chromatography by going to this page archived from the Colorado University site. Linking to other sites is always a little bit hazardous because sites change.
If you find that this link doesn't work, please contact me via the address on the About this site page. If this is the first set of questions you have done, please read the introductory page before you start.
Use the BACK button on your browser to return quickly to this page. Carrying out column chromatography The column In thin layer chromatography, the stationary phase is a thin layer of silica gel or alumina on a glass, metal or plastic plate. Using the column Suppose you wanted to separate a mixture of two coloured compounds - one yellow, one blue. 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.
The next set of diagrams shows what might happen over time. Explaining what is happening This assumes that you have read the explanation for what happens during thin layer chromatography. What if you want to collect the blue compound as well? At Pitt, alumina is used to pack the column and provides the stationary phase upon which the sample adsorbs. Adsorption is the process of molecules 'adhering' to one another, without the making of chemical bonds. Molecules in the sample will desorb off the adsorbent and dissolve in the eluent when the polarity of the eluent matches the polarity of the molecules.
If a sample mixture contains just two compounds, one polar and the other nonpolar, then the elution process would begin by adding a nonpolar eluent or solvent.
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