I have no experience with column chromatography but I have been reading protocols and watching videos online that explain the principles and procedures. When you are running the column how do you know when one fraction has stopped coming out of the column and the next one has begun? Is there a visual color difference? Does the mobile phase flow stop between fractions? Is it based on time?
More specifically a protocol on how to separate a solution indicated that the substance of interest will be in fraction one. How do I know when to stop collecting the first fraction?
Dear John,
I evaluate odd collecting tubes with TLC, and observed under UV lamp that way, I'm collecting tubes containing the same generating different fractions. Sometimes it is shown in column solor changes naked eye, but not always.
Regards!!
Dear John,
I evaluate odd collecting tubes with TLC, and observed under UV lamp that way, I'm collecting tubes containing the same generating different fractions. Sometimes it is shown in column solor changes naked eye, but not always.
Regards!!
The key is having a way to detect your sample of interest. If you have an online detector, such as UV-visible spectroscopy, and your compound absorbs in that wavelength range, then you can monitor its elution in real time. If not, then you will have to collect fractions over a period of time, then analyze those fractions for your compound(s) of interest. Many people use colorimetric assays, TLC, MS, etc. Practically anyone over in the Biochem Dept at ISU could show you how.
If you developed your column method by TLC then you can determine the number of column volumes required to elute each compound. To do this measure the solvent migration distance on the TLC plate and the migration distance for the compounds or spots on the plate that are of interest to you. Divide the spot distance by the solvent migration distance to determine the compound's Rf value. You can calculate the number of column volumes (CV) required to elute each compound from the reciprocal of the Rf (e.g. 1/Rf). This works if your method is isocratic (constant solvent ratio during the separation).
You can calculate the column volume for your column using the equation pi*r2*L*0.7.
Once you know your column's volume, in mL, then you can collect fractions based on expected elution volume. For example, if one of the compounds you want to collect has an Rf of 0.5, the compound will elute in 2 CV. If another has an Rf value of 0.2 it will elute in 5 CV.
A resource you may want to check is www.flash-purification.com. It has a lot of information related to column chromatography.
Thank you everyone, all of your answers were extremely helpful.
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