5 Ways to Make Size Exclusion Chromatography A More Approachable Topic

Introduction: Size Exclusion Chromatography

Natural products from different sources like plants, animals, fungi, bacteria, etc are a potpourri of different chemical substances. None of the metabolites or macro/macromolecules is produced by biological organisms as a single substance or metabolite. For their utilization, analysis, and chemical prospection, the separation of these complex metabolites becomes important.

There are different types of separation techniques that are exploited as per the requirement in both research and industrial fields. Chromatography techniques are one of them. They rely on the ‘differential affinities of different compounds in a mixture for 2 phases of the chromatogram’; stationary and mobile phases. If compound A has a higher affinity for a mobile phase, it will move faster, travel longer distances, and/or elute faster. There are different types of chromatography techniques, one of which is size exclusion chromatography.

Size exclusion chromatography (SEC) is a liquid chromatography technique in which the mobile phase is liquid and the stationary phase is a polymer matrix. The stationary phase support in SEC is a column meaning that the stationary phase is packed into a vertical column while the mobile phase flows through it. 

SEC finds abundant usage in protein mixture fractionation experiments and fractionation of several other water-soluble polymers. It is widely used for industrial polymers, macromolecules like nucleic acids (DNA/RNA), proteins, antibodies, and enzymes where the sizes of the individual components of the mixture are relatively larger.

While SEC enjoys a good repo in both academic research and industry, students often stumble when they are first introduced to this separation technique. The dilemma of how to comprehend the details while practically applying the basic idea behind SEC makes them go wary and tense.

For teachers explaining SEC in their classes, this article can provide real help as it highlights the blocks encountered by students and lists practical solutions to resolve them. By the end, we’ll share why a virtual lab simulation will prove useful not only for your students but also for you as an educator to deliver concepts more efficiently.

3 reasons why Size Exclusion Chromatography can be tricky to teach or learn

There are 3 reasons why students dread and get confused about the topic of the Size Exclusion Chromatography separation technique. Acknowledging these blocks is the first step toward making the topic more approachable. 

1. Biologists fear chemistry.

It is one of the most common reasons that teachers delivering lessons about separation techniques are oblivious of. The majority of biology students that study methodologies that incorporate fundamental chemical ideas fear the subject. Since analytical techniques like SEC and even other chromatographic techniques work at the junction of different chemistry concepts, biology students can be unnerved at the mere introduction of these concepts. Lack of idea about different phases of SEC, its underlying principle, column packaging technique, and different types of polymers used in packaging (porous glasses, silica, agarose, dextran polymers, polyacrylamide, etc) are some intimidating topics of SEC for students.

2. Lack of both practical handling and visualization.

Most of the time, high school and college students don't get enough practice handling lab equipment. These kinds of studies are typically carried out in groups or demonstrated by educators themselves in labs due to a lack of funding or consumables. In this situation, learning the method becomes quite passive where students don’t know how to load the sample, how cautiously one should handle the SEC apparatus, or how one should select an appropriate size exclusion column. Additionally, the deficiency of any video demonstrations of the SEC also shuns any possibility of learning these minute details and tricks more interactively. No wonder why theoretical lessons on SEC can become non-conductive.

3. Inability to distinguish between different chromatography techniques.

Since there are several chromatography techniques, students often fail to understand the differences between each one of them. And the case of the SEC is no different. With little or no information about how to objectively approach the problem, students are often seen rote learning the topic of chromatography and dumping the idea once their exams end. Though the SEC technique has a wide range of applicability and potential, ignorance and lack of knowledge leave them under-utilized.

5 ways to make Size Exclusion Chromatography a more approachable topic to understand

To address the blocks encountered while teaching the Size Exclusion Chromatography technique, educators can engage the under-listed solutions in their classes. These can clarify many instrumental aspects of SEC. Not only can they make teaching easier for educators like you but they will also make lessons clearer and easier to assimilate for your students.

1. Drive out the fear of chemistry 

Since SEC involves terminologies and concepts that can be overwhelming at first, easing out their meanings for your students can serve the purpose of slashing out the fear of chemistry. Further explaining about different chemistries of different chromatography techniques can help. We list a few tips and tricks that you can put to use in your next class.

• Explaining the basic principle behind SEC: The different components of a mixture are separated by the technique of size exclusion chromatography exclusively based on their size. SEC is a type of liquid chromatography where the molecules in the sample are separated according to their sizes. The stationary phase is built on columnar support and the separation process is supported by gravity. You can explain the concept of the stationary phase of SEC as a “trap for smaller molecules”. The smaller molecules of the sample mixture diffuse inside the spherical porous beads of the stationary phase of SEC which retards their speed while the larger molecules of the sample mixture pass easily and faster down the column.  

Figure: Illustration explaining the underlying principle of SEC. Larger molecules pass through the interstitial spaces between the porous beads. Smaller molecules get trapped inside the porous beads and their movement is thus slowed down. Image Source

• Explaining the different types of chromatography: There are several different types of chromatography like paper chromatography, thin layer chromatography (TLC), ion exchange chromatography, etc. Explain the different stationary and mobile phases in each. This will help your students understand the rationale behind their work. This will also help them in making the correct choice of the technique according to their hypothesis and work requirements. You can use TLC (Thin Layer Chromatography) simulation from Labster too.

Figure: Stationary and mobile phases in different types of chromatography. Image Source

2. Reason out the intricacies of SEC

While selecting different phases of SEC, several intricacies are involved. Making appropriate choices of mobile phase, stationary phase and its composition, length, etc are some result deciding factors. Hence, explaining their importance to students can illuminate the dark spaces of SEC feared by students. 

• Selecting appropriate bead size of size exclusion column: Explaining the importance of the size exclusion column can make the technique clearer to your students. When we have a given sample mixture at hand, the selection of the size exclusion column depends on the ‘2 extreme size limits of molecules in the sample mixture. For instance, if the size of the smallest molecule in the sample mixture is 45µm, then the smallest bead size should be at least 45µm so that the bigger molecules pass faster while the 45µm molecules diffuse into the bead and their speed is retarded. This will ensure that the larger molecule moves past and gets separated from the smaller one.

• Selecting appropriate dimensions of size exclusion column: The “resolution” of the SEC technique improvises with the increase in column length. So, if you are looking for a fine resolution of sample mixture in SEC, the use of a longer size exclusion column is recommended. The “sample loading capacity” in SEC improvises with the increase in column diameter. So, if you are planning to load a large number of samples, the use of a gel with a greater diameter is recommended.   

Similarly, try to explain the reasons behind each of the intricacies involved in SEC experiments. This can make your student understand how research work demands unwavering attention and dedication and prepares the next generation of scientists for STEM research.

3. Try to maximize lab handling practices or otherwise involve visualization sessions

Since SEC or any other technique for that matter is best taught by the physical involvement of students in the class, educators and teachers can try to approach Sci-tech-based companies to source some extra consumables from them. Usually, the industry waste (obviously check for hazards) can be recycled and used for lab demonstrations. Since schools and colleges aren’t funded as good as industries, building collaboration with industries can help the former in a very big way.

If the lab handling is still compromised, educators can opt for interactive YouTube videos if available. The real-time run of SEC can make the topic intriguing and interactive for classroom teaching. Alternatively, educators can use the Size Exclusion Chromatography simulation provided by Labster.

4. Cite practical uses of SEC

Size exclusion chromatography is used for many purposes in both research and industry. We are providing a few examples that you can cite for your students. This will help them recognize the practical applicability of learning the technique. 

• For protein fractionation

• For nucleic acid fractionation (DNA/RNA)

• For separating different types of enzymes

• For separating different types of water-soluble polymers

• For separating different types of antibodies

• For fractionation of industrial polymers

• For purification of sugars, viruses, hormones

• For examination of natural organic matter present in water (stability and properties)

Figure: A snippet from Size Exclusion Chromatography simulation by Labster showing monomers and oligomers. It is available for University / College classes. 

5. Use virtual lab simulations

Since SEC can be too theoretical in the absence of lab handling sessions, educators are advised to search for visually interactive videos. YouTube doesn’t always provide such an option for all the topics. To overcome this visualization constraint in the learning process of the students, virtual laboratory simulations from Labster provide teachers with an option to use the Size Exclusion Chromatography simulation. 

This can help in making more insightful points as students are rendered with better visual options. Your students don’t have to struggle to imagine different steps of SEC themselves as our interactive simulation along with gamification elements comes to the rescue. By using this way of active and immersive teaching, our virtual learning platform takes an advent in the field of Science to make the upcoming scientists thorough with the “basics of their respective subjects”. 

You can learn more about the Size Exclusion Chromatography simulation here or get in touch to find out how you can start using virtual labs with your students.

Figure: A snippet from Size Exclusion Chromatography simulation by Labster showing the Sec experiment and chromatogram in a virtual lab. It is available for University / College classes.