What is size exclusion chromatography?
Size exclusion chromatography (SEC), also known as gel filtration, is the mildest of all the chromatography techniques. SEC separates molecules by differences in size as they pass through a resin packed in a column. Unlike techniques such as ion exchange chromatography (IEX) or affinity chromatography (AC), molecules do not bind to the chromatography resin, which means that buffer composition does not directly affect resolution (i.e., the degree of separation between peaks). Consequently, an advantage of SEC is that conditions can be varied to suit the type of sample or the requirements for further purification, analysis, or storage without altering the separation. In this article, we highlight why column selection is critical for improving both efficiency and data quality in SEC workflows. By bringing together SEC fundamentals, practical guidance on column and resin choice, and comparative performance data, the article shows how high resolution separations can be achieved without extending run times. The goal is to help researchers make informed decisions that reduce reruns, streamline purification steps, and support more confident progress from routine analysis through small scale preparative protein purification.
Figure 1. Size exclusion chromatography (SEC)
How does size exclusion chromatography work?
SEC resins consist of a porous matrix of spherical particles (beads) that lack reactivity and adsorptive properties. After a sample has entered the column, molecules larger than the pores are unable to diffuse into the beads, so they elute first. Molecules that range in size between the very big and very small can penetrate the pores to varying degrees based on their size. If a molecule is smaller than the smallest of the pores in the resin, then it will be able to enter the total pore volume. Molecules that enter the total pore volume are eluted last. Samples are eluted isocratically so there is no need to use different buffers during the separation.
Choose the right column
In research, every minute counts. Whether you’re pushing the boundaries of science or simply planning your next big idea, time spent waiting for results is time lost. Choosing the right SEC column can make all the difference, not just in resolution, but in how efficiently you move forward with your work.
Particle size, column dimension, minimizing system volumes, flow rate, and the packing of the column are all important factors for you to consider to achieving high resolution peaks. The selectivity of a resin, or the distance between two peaks, together with the efficiency affects the resolution between peaks. The selectivity of a resin depends on the properties of the resin such as pore size distribution, the interactions between sample and resin, and the conditions used. The efficiency of a resin depends mainly on particle size, the column format, and the packing of the column.
Each of these areas are covered in even greater detail in the Size Exclusion Chromatography Handbook.
When to use preparative SEC for protein purification
Preparative SEC is a high-resolution size-based separation of biomolecules with collection fractionation for further analysis or scale-up. Preparative SEC is performed to isolate one or more components of a sample. Separated components can be directly transferred to a suitable buffer for assay or storage. Sample volumes of 0.5 to 4% of the total column volume are applied at low flow rates using long columns, often 60 cm or longer. Because separation occurs within a single column volume (CV), optimal column packing is essential. For convenience, consistency, and performance, prepacked SEC columns offer clear advantages.
Data behind the decision: Choosing resins and prepacked columns
Efficient column packing is essential in preparative SEC, particularly for high-resolution separations. Depending on the resolution and yield needed, column bed heights of up to 100 cm are recommended for preparative SEC separation with resin bead sizes of approximately 9 to 34 µm (see Table 1). The required bed volume and hence the diameter of the column is determined by the sample volume.
Table 1 consolidates key performance and operating characteristics of commonly used preparative SEC resins into a single, easy to compare format, helping readers translate theory into practical column selection decisions. By showing differences in efficiency, operational and cleaning in place pH ranges, and particle size, the table clarifies how resin properties directly influence resolution, robustness, and suitability for specific applications. This context reinforces the article’s core message that achieving high quality, reliable SEC separations depends not only on method conditions, but also on informed selection of the appropriate resin and column format for the intended workflow.
Table 1. Characteristics of resins used for preparative SEC purifications
| Product Efficiency1 | Operational2 | Cleaning-in-place (CIP) pH3 | Particle size, d50v (µm)4 |
|---|---|---|---|
| Superdex™ 30 Increase | > 43 000 | 3 to 12 1 to 14 | 9 |
| Superdex™ 75 Increase | > 43 000 | 3 to 12 1 to 14 | 9 |
| Superdex™ 200 Increase | > 48 000 | 3 to 12 1 to 14 | 8.6 |
| Superdex™ 30 prep grade | > 13 000 | 3 to 12 1 to 14 | 34 |
| Superdex™ 75 prep grade | > 13 000 | 3 to 12 1 to 14 | 34 |
| Superdex™ 200 prep grade | > 13 000 | 3 to 12 1 to 14 | 34 |
1Theoretical plates per meter (prepacked columns only).
2pH range where resin can be operated without significant change in function.
3pH range where resin can be subjected to cleaning- or sanitization-in-place without significant change in function.
4Approximate median particle size of the cumulative volume distribution.
A well-packed column should produce narrow symmetrical peaks with minimal peak broadening. The uniformity of the packed bed and the particles influence the flow through the column and affects the shape and width of the peaks. High-performance SEC resins with high bed uniformity, which are smaller with more uniform particles give decreased peak widths and improved resolution.
Scientists often encounter the challenge of achieving reliable, high resolution SEC separations without sacrificing time or productivity. In many protein purification workflows, insufficient resolution can lead to overlapping peaks, uncertainty in results, and repeated runs that slow progress and consume valuable resources. At the same time, extended run times delay decisions and downstream work. Our recent study speaks directly to that challenge by demonstrating how the right SEC column can deliver clean, high confidence separations more efficiently, which helps researchers reduce reruns, limit troubleshooting, and keep their work moving forward without compromise.
A benchmark study of small-scale preparative SEC (sample volumes < 0.5 mL) comparing performance across commonly used SEC columns on an ÄKTA pure™ 25 chromatography system, with optimized configuration. Small capillary (0.25 mm ID) was used and column positioned directly in UV monitor to get optimal results.(1)
Figure 2 shows a resolution comparison between Cytiva Superdex™ 75 Increase columns and Bio-Rad ENrich™ SEC 70 columns across the tested samples. The data demonstrate that the Superdex™ 75 Increase column delivers consistently strong separation performance, producing narrower, better defined peaks under the benchmark conditions used. Higher resolution at comparable operating conditions translates into cleaner separations, helping reduce ambiguity in peak identification and minimizing the need for repeat runs when analyzing complex protein mixtures
Figure 2. The comparison shows a 30% higher resolution using the Cytiva Superdex™ 75 Increase columns at the same flowrate, or half the run time to receive a similar resolution. (2) (3)
Figure 3 presents a similar resolution comparison for larger range separations, evaluating Cytiva Superdex™ 200 Increase columns against Bio-Rad ENrich™ SEC 650 columns. Here, the data show that Superdex™ 200 Increase achieves higher resolution at the same flow rate, indicating improved efficiency without extending run time. This performance advantage supports more reliable separations for larger proteins and biomolecular assemblies, enabling confident decision making while saving time through fewer reruns and more predictable SEC workflows.
Figure 3. Resolution comparison data between Cytiva Superdex™ 200 Increase columns and Bio-Rad ENrich™ SEC 650 columns. The comparison shows a 60% higher resolution using the Cytiva Superdex™ 200 Increase columns at the same flowrate, or half the run time to receive a similar resolution. (3)
Figures 2 and 3 are central to the comparator study because they provide direct, experimental evidence comparing the separation performance of Cytiva Superdex™ Increase columns with Bio-Rad ENrich™ SEC columns under the same benchmark conditions. Together, the figures show that Superdex™ 75 Increase and Superdex™ 200 Increase deliver higher resolution and more efficient separations across different molecular weight ranges, supporting cleaner peak separation, fewer reruns, and more reliable data. These specific products were selected because they represent commonly used, functionally comparable SEC columns targeted at similar applications—small scale preparative SEC for proteins—making the comparison relevant to scientists choosing between established options on the market. By evaluating matched column types across two size ranges, the study demonstrates that the observed performance advantages are consistent and application relevant, strengthening the conclusion that column choice can directly impact resolution, run time, and overall workflow efficiency.
Figures 2 and 3 present comparative resolution data obtained under defined, optimized experimental conditions using selected SEC column formats tested on a single chromatography system configuration. The results are intended to illustrate relative resolution performance within the scope of the study design and for the specific samples, flow rates, and column dimensions evaluated. These data do not account for all variables encountered in routine laboratory workflows, including differences in systems, methods, sample types, or operating conditions, and should therefore be considered comparative benchmarks rather than predictive of performance across all applications.
Looking at the results (see Fig. 3), the Superdex™ 200 Increase column consistently delivers high resolution across all tested samples. This means fewer runs, less troubleshooting, and more confidence in your data. Higher resolution translates to cleaner separations, which reduces repeat experiments and saves time.
You don’t have to trade resolution for speed in SEC. By choosing the right SEC column—particularly high‑performance, well‑packed prepacked columns like Superdex™ Increase—scientists can achieve higher resolution separations in less time, reduce reruns, and move research forward faster. In short, the right SEC column delivers cleaner data, fewer repeats, and more efficient workflows, allowing you to focus on science rather than waiting on chromatography runs.
In summary, these are the shown advantages of choosing size exclusion columns from Cytiva, compared with Bio-Rad ENrich™ size exclusion columns:
- Cytiva Superdex™ 200 Increase and Cytiva Superdex™ 75 Increase offers half the run time at similar resolution, compared with the equivalent Bio-Rad ENrich™ columns.
- An up to 60% higher resolution with Superdex™ 200 Increase at same flow rate is received.
Optimizing your separation
The success of size exclusion chromatography run depends primarily on choosing conditions that give sufficient selectivity and counteract peak broadening effects during the separation. Prepacked columns are delivered with recommended running conditions that give satisfactory results in most situations, but optimization might sometimes be necessary to reach the required resolution. As mentioned in How does size exclusion chromatography work, resolution is a function of the selectivity of the resin and the efficiency of that resin to produce narrow peaks (minimal peak broadening).
If optimization is needed, follow these steps (given in order of priority):
- Select a resin with a suitable fractionation range providing optimal resolution.
- Select a column with a bed height providing the required resolution. A bed height between 30 and 100 cm is recommended for preparative separation.
- Select a size column appropriate for the volume of sample that needs to be processed.
- Select the highest flow rate that maintains resolution and minimizes separation time.
Key characteristics of SEC resins used for preparative SEC
For scientists looking to build on the fundamentals covered in this article, these resources offer practical support and deeper insight into chromatography and protein purification. They include guidance on SEC running conditions, access to tools and communities designed to support method development and system configuration, and learning opportunities that span core chromatography techniques—helping scientists apply best practices, optimize workflows, and confidently advance their purification strategies.
For deeper insight into chromatography and protein purification, explore our helpful resources on chromatography fundamentals, method development guidance, and application-focused content:
- Join the Purify Club
- Explore the Purify App for configuring and exploring ÄKTA™ chromatography systems, columns, and resins to support your protein purification needs.
- Discover our free eLearning course that guides you through a range of chromatography techniques.
Conclusion
Choosing the right SEC column is critical to achieving high resolution separations without compromising run time. By demonstrating how high performance SEC columns can deliver cleaner separations more efficiently, this article highlights how thoughtful column selection helps reduce reruns, streamline workflows, and keep research moving forward with confidence.
References
- Optimal configuration of ÄKTA pure™ 25 for small-scale SEC, as standard configuration of ÄKTA pure™ 25 would not provide optimal results.
- Studies perform at Cytiva Uppsala. Data held at Cytiva, Uppsala. The information presented is based on benchmarking studies performed at Cytiva, Uppsala between November 2015, February 2016 and November 2025. The purifications were performed in triplicate, using recommended protocol from the suppliers. The data used to support this study can be made available upon request to cytiva.com/contact.
- Bio-Rad ENrich™ SEC 70 and Bio-Rad ENrich™ SEC 650 are trademarks of Bio-Rad Laboratories, Inc.