Column chromatography is a common technique used to separate individual compounds from a mixture. You can use column chromatography on both a small or a large scale to isolate and purify material for use in a later experiment.
If you are new to column chromatography or just want a refresher, then read on for a quick description of the basic process of column chromatography setup and running.
Grab a Chromatography Column
A chromatography column is a glass or plastic tube that you set vertically and fill with a stationary phase.
What size should I choose?
You can use different-sized columns to separate samples in amounts anywhere from micrograms to kilograms.
The column’s diameter is directly related to the scale of your sample, and the length depends on the difficulty of separation.
Though numbers can vary from column to column, just to give you an idea, a good starting point is to keep the mass of your crude sample somewhere between 1 to 5% of the total mass of the stationary phase. And keep that mass percentage on the lower side if you predict a difficult separation.
Also, do your best to select the smallest and shortest possible column that gives you a good separation. This way you can save time, packing material, and eluent.
Pick a Stationary Phase
Like in all other chromatography methods, column chromatography separates the components of a mixture based on their properties and how they interact with the stationary phase and/or mobile phase.
The stationary phase is a matrix or resin with functional groups that interact with your sample. Because molecules vary greatly in their properties, you can exploit different types of interactions to separate compounds.
For example, you can select a column to do separations according to a molecule’s hydrophobicity (hydrophobic interaction column), polarity (silica column), or electrostatic charge (ion-exchange column).
Do Some Packing
Column packing in chromatography is a critical step in your separation procedure. A poorly packed column can lead to uneven flow and band broadening, both of which give rise to poor separation.
Dry vs. Wet Packing
There are two common ways you can fill a chromatography column:
1. Dry Packing
For this method, add your dry solid phase to the column and pass equilibria buffer or starting solvent to saturate the solid.
2. Wet Packing
Here, you mix both liquid and solid outside of the column and pour it into the column.
I personally prefer wet packing because it’s usually faster and seems to use up less liquid.
Regardless of how you fill the column, always make sure that there are no bubbles and that the stationary bed is even.
Prep the Eluent
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. But if you come across a sample that isn’t easily purified with an eluent of constant composition, then switch to a gradient run. You’ll have better control of the separation as the mobile phase conditions (pH, salinity, or polarity) gradually change from low to high eluting strength.
Load and Run Your Chromatography Column
There are a few key points when loading and running your column chromatography experiment:
- Do not load your sample until you have all your materials gathered and set up. So, plan ahead. Only when you are ready to run the column, load your sample by pipetting it onto the packed solid bed.
- Once the sample is loaded, pour in the eluent and start the flow immediately.
- Do not let the column dry out and do not stop in the middle of the run. When your sample is adsorbed onto the resin, the components will dissolve in the running liquid and the separation will start. Any disruptions in the partitioning equilibrium will mess up your resolution.
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). Remember that it’s because of these differences in elution speed that the components of a mixture separate.
When to Start Your Collection
I always find it safer to collect fractions from the start of the chromatography column run—better safe than sorry I guess. But if you’re starting with a wash and you are sure that your desired material will not elute, then collect that wash as one big fraction. After that, collect smaller fractions when you begin eluting your material.
The size of fractions is typically about 10 to 20% of the column volume. But it can be tricky to decide because the fraction size doesn’t only depend on the size of the column, but also on the difficulty of separation.
Keep in mind that collecting more and smaller fractions may give you more chances of isolating your material free of contaminants. Yet, don’t go too small because you’ll needlessly end up with a lot more fractions to analyze.
Find Your Stuff
You only need a very small amount from each fraction to figure out where your material of interest ended up. The method that you use to analyze your fractions will depend on the type of molecule you’re working on.
If you isolate protein or DNA, run a gel to identify the fractions that have it. Or do an activity assay if you’re working with an enzyme. Alternatively, run a TLC plate for plant extracts or lab-made chemical compounds. Other methods are available, and I would say to go with what’s more prevalent in the literature for your type of molecule.
Finally, the screening of fractions should also give you information on the purity of your material. Use this information to pool the fractions with the highest purity to obtain a yield and continue on to your next experiment.
Want to Save Time?
The process I described here is geared for running columns manually—or how I like to call them: hand-columns. But well-funded labs may use automated systems such as fast protein liquid chromatography or flash column chromatography.
They work the same way except that there’s a lot less work for you to do. A pump, detector, and fraction collector cover most of the process. You simply set up a pre-packed chromatography column (yes, you can buy them!), select the elution conditions, hit “start”, and go do something else while it runs.
Automated systems can rock your world by saving you a lot of time. But I still recommend learning how to do hand-columns. There’s nothing like having the practice of doing it yourself to know how to tackle issues and troubleshoot a procedure.
So, there you have it—now go run your column!
In my next article, I discuss some of the different types of stationary phases that you can use to clean up your samples.
Originally published August 2016. Reviewed and updated September 2021.