by: Suzy Wu
When I entered college as a chemical engineering student, I had barely any lab experience. I had always envisioned university research labs as cutting-edge and far out of reach, but after working as a research assistant for Gebbie Lab, the previously overwhelming equipment and techniques do not seem so intimidating any more. In fact, they boil down to quite simple mechanisms. For those of you who are curious about the methods and apparatus we use in lab, I hope to give you a brief introduction via this blog post. To start with, let’s focus on a basic separation method often encountered in general chemistry lab — column chromatography.
Separation of a mixture yields different colored bands in the column
What is column chromatography?
In general, chromatography is a purification and separation technique to separate chemical compounds from a mixture based on different adsorptivities or polarities6. Common types of chromatography methods include Thin-Layer Chromatography (TLC), Gas Chromatography (GC), Liquid Column Chromatography (our focus of today), Size Exclusion/Ion Exchange/Affinity Chromatography and so on.
Specifically, column chromatography is easy to perform without complex training or post-analysis. A column is a glass apparatus through which the liquid flow rate is controlled by a stopcock: maximum flow when aligned vertical to column, and zero flow when aligned horizontally. The mechanism of column chromatography is that the different chemical substances in a mixture interact to the silica or alumina gel in the column (stationary phase) at different extents when dissolved in the liquid solvent (mobile phase) and poured into column1. In a common setting, chemicals are deposited down a column by gravity. The chemical compounds move at varying speeds through the column, forming different bands of chemicals for a colored sample. The compounds which interact more strongly run more slowly and form the upper layer, whereas the ones that have weaker adsorption run faster to form the lower layer. This separation of bands allows for separation of the chemicals, via collecting the eluted molecules with corresponding flasks.
Mechanism of separation using column chromatography Image Source: Khan Academy
How to build and use a column?
A peak inside a column. Image Source: BiteSizeBio
Now that we are familiar with the principles, it is not hard to prepare a column for use. To start off, let’s gather the following materials: a glass column with stopcock, collecting flask or beaker, glass wool to tamp down column, silicon or aluminum oxide particles (silica or alumina for short, on the scale of μm in size), and liquid solvent to dissolve and elute desired molecules. With the ingredients in hand, we can follow this recipe:
- Pack column with cotton or glass wool to block the stationary phase from getting flushed down the column
- Prepare silica or alumina slurry and let the particles activate
- Pour slurry into the column, let settle, and drain liquid to ~0.5cm above the solid level
- Transfer dissolved mixture into the column, let settle, and collect bands of chemicals with flasks or beakers
- Flush column with solvent to remove residual chemicals within column
Usage in the lab
In many research labs, chromatography is used to isolate different molecules. In Gebbie Lab, this technique is often used for purification following synthesis. After purifying the mixture with charcoal filtration, an alumina column is used to remove remaining activated charcoal or other impurities. Further purification and separation methods then isolate out the desired product. The column is an indispensable step because the fine stationary phase blocks particulates that are otherwise difficult to remove via filtration. Despite the simple principle, there are several things to be aware of when running a column.
- Tamp down glass wool tightly and avoid forming air gaps in column – otherwise liquid will drain very slowly
- Don’t let column run dry – otherwise stationary phase will crack and disrupt layers
- Make sure liquid level is even, not tilted; for this purpose, consider transferring dissolved sample/mixture with a pipette
- Let column drain out and properly dispose column resins into solid waste
Column chromatography performed in the Gebbie Lab
Beyond usage in research labs, column chromatography can also be applied in large scales for analysis and separation in the fields of organic chemistry, pharmaceuticals, environmental studies, and food science. A higher-resolution method is called High-Performance Liquid Chromatography (HPLC), which utilizes finer adsorbent particles and applies high pressure rather than gravity to elude mobile phase down the column7, but it shares the same principles as a column.
If you’re interested in learning more, here are some useful (and free!) resources to check out:
https://www.khanacademy.org/science/class-11-chemistry-india/xfbb6cb8fc2bd00c8:in-in-organic-chemistry-some-basic-principles-and-techniques/xfbb6cb8fc2bd00c8:in-in-methods-of-purification-of-organic-compounds/v/column-chromatography — Lectures and videos about the basics and different types of chromatography from Khan Academy
https://www.youtube.com/watch?v=yig3QCfBTzc – A detailed introduction to the procedures of column chromatography and post-analysis techniques
- Guerrero, Angela. “Principles of chromatography.” Khan Academy. Accessed 31 July 2021. https://www.khanacademy.org/science/class-11-chemistry-india/xfbb6cb8fc2bd00c8:in-in-organic-chemistry-some-basic-principles-and-techniques/xfbb6cb8fc2bd00c8:in-in-methods-of-purification-of-organic-compounds/a/principles-of-chromatography.
- Parichha, Arpan. “HPLC || High Performance Liquid Chromatography.” YouTube, uploaded by Animated biology with arpan, 16 Sept. 2020, https://www.youtube.com/watch?v=Vr5t-cgHHG4.
- Thongdumhyu, Rattiya. “Column chromatography chemistry in the lab.” Shutterstock, https://www.shutterstock.com/zh/image-photo/column-chromatography-chemistry-lab-470534546. Accessed 1 Aug. 2021.
- Torres, Jessica. “The Basics of Running a Chromatography Column.” BiteSizeBio, 15 Aug. 2016. https://bitesizebio.com/29947/basics-chromatography-column/. Accessed 31 July 2021.
- Vladyslav, Sodel. “The laboratory scientist prepares samples for download to High-Performance Chromatograph Mass Spectrometry.” Shutterstock, https://www.shutterstock.com/zh/image-photo/laboratory-scientist-prepares-samples-download-highperformance-1503327674. Accessed 1 Aug. 2021.
- Wikipedia Contributors. “Column chromatography.” Wikipedia, Wikimedia Foundation, 5 Dec. 2020, https://en.wikipedia.org/wiki/Column_chromatography. Accessed 31 July 2021.
- Wikipedia Contributors. “High-performance liquid chromatography.” Wikipedia, Wikimedia Foundation, 26 May 2021, https://en.wikipedia.org/wiki/High-performance_liquid_chromatography. Accessed 1 Aug. 2021.