Configuring the Best GPC/SEC Column Set for Your Sample

If you have worked with GPC/SEC (e.g., OMNISEC), you will know that all analyses rely on the configuration of the column set. Selecting the right columns for your application can sometimes be challenging. Occasionally, a completely new column set configuration may be needed for different types of samples.

There are several considerations to keep in mind when selecting the best column set for your sample. Considerations include the molecular size/weight range, compatibility of the mobile phase needed to dissolve the sample with the columns, and the compatibility of functional groups present in the sample with the stationary phase of the columns. If using multiple columns, consider whether they complement each other and in what order they should be connected.

This post will provide general information on columns and offer insights on how to create a suitable column set for your sample. The information here primarily concerns selecting the best column set from a molecular size/weight range perspective. For information on columns available for various mobile phase analyses, please refer to the linked post here and webinar.

What Happens in a Column Set?

Let’s start with a brief overview of how separation works within a GPC/SEC column set. As seen in the image above, a dissolved mixture of two samples of different sizes is introduced into the column. Over time, the two sample fractions and the solvent move through the column at different rates. The largest sample elutes first (Population 2), followed by the smaller sample (Population 1), and finally the solvent elutes.

It is important to understand that separation is based on the molecular size of the sample, not the molecular weight!

This separation occurs because the stationary phase of the column is composed of gel particles with pores. These pores are large enough for the sample molecules to diffuse into them as they pass through the column. Small molecules diffuse into the pores more easily, spending more time in the pores than medium and large molecules. The largest molecules do not easily diffuse into the pores, thus taking a simpler and faster path through the column.

With GPC/SEC, separations are based on molecular size, with large molecules eluting first followed by medium and small molecules. Please refer to the animated depiction below for this process. The large triangle elutes first without spending much time diffusing into the pores, followed by the medium square, and finally, the small circle can easily move into the pores.

Mixed Bed and Single Pore Size Columns

The differences between these two types of columns are detailed in this post. In short, mixed bed columns contain stationary phase gel with a mix of particle and pore sizes, providing resolution over a wide range of molecular sizes/weights. Single pore size columns contain a homogeneous stationary phase gel, offering superior resolution over a relatively limited molecular size/weight range.

Creating a Column Set by Combining Columns

To create an optimized column set for your sample, you will need to combine two or more columns. (However, if a single column provides sufficient resolution for your sample and is all you need, then no column combination is necessary.) This will save time and mobile phase. This is often the case for protein samples. While the molecular weight of proteins spans a wide range, the size range of protein samples is narrower than for polymers due to their folded structures.

There are three common approaches to take when combining columns to create a set. We suggest these approaches, but please note that they are not exhaustive.

2 Mixed Bed Columns: The most common general-purpose column set encompassing organic, aqueous, and specialty columns. Specifically, two of the same mixed bed columns are used. For instance, two A6000M columns, which are mixed bed columns for aqueous analysis, are commonly used for analyzing batches of polysaccharides. Using 2 mixed bed columns like this tends to maximize molecular size/weight range and reach the ideal point where run times are kept to about 45 minutes or less.

Combining High/Low MW Columns with a Mixed Bed Column: For samples where there are especially high or low molecular size/weight species present, such as aggregates or oligomers, you may prefer to add a high/low single pore size column to a single mixed bed column. The high/low molecular weight single pore size columns ensure sufficient resolution for specific features of the sample at one extreme of the molecular size continuum, while the mixed bed column accounts for the remaining sample material. If you wish to improve the resolution between the sample and the solvent peak, add a low molecular weight column to the mixed bed column.

Combining Multiple Single Pore Size Columns: This strategy is suitable when you know that the full molecular size/weight range resolution provided by mixed bed columns is not necessary. This type of column set may be advantageous in environments monitoring similar samples like QC. The benefit is that you can gain maximum resolution at the molecular size/weight of interest to the sample associated. You might combine two or more single pore size columns to cover a broad range of molecular sizes/weights, like T5000 + T3000 + T1000. In this example, there is an extended analysis time but enhanced resolution.

When combining single pore size columns, it is important to ensure that columns are not more than one column step apart. This will prevent differences in the resolution range from occurring. Referring to the example of three single pore size columns above, simply combining T5000 + T1000 is not recommended. The difference in resolution range can create elution profiles causing odd peak shapes and artifacts in your data as shown below, resulting from chromatography rather than the sample itself.

Order of Columns

Many people wonder, ‘In what order should the columns be connected when combining them?’

The sensible order we recommend is to start with the column of highest molecular size/weight and gradually move to the column of lowest molecular size/weight. This allows the largest molecules to begin separating as the whole sample mixture is first introduced to the column set. If the columns are arranged in reverse order, the largest molecules must squeeze through the low molecular size/weight column, potentially obstructing smaller molecules from diffusing into pores.

Thus, in the earlier example, we used the T5000 + T3000 + T1000 columns. When combining single pore size columns with mixed bed columns, add the high molecular size/weight single pore size column before the mixed bed column and the low molecular size/weight single pore size column after the mixed bed column.