ExcsepTM SEC Columns User Manual

Introduction 

ExcsepTM SEC is a high performance liquid chromatography column based on the volume exclusion separation mechanism. Its packing material is made of ultra-high mechanical strength, high pore volume silica gel microspheres, combined with unique surface hydrophilic chemical modification, which provides excellent chromatographic separation performance and is suitable for the separation of water-soluble molecules such as polysaccharides, as well as biological macromolecules such as peptides, biological enzymes, antibodies/proteins, and other biomolecules.

Size exclusion chromatography (SEC) is a molecular sieve chromatography mode, also known as gel exclusion chromatography. It separates analytes mainly according to their molecular weight (size): high molecular weight components cannot enter the pores of the packing material in the chromatography and elute first; medium molecular weight groups can enter some of the stationary phase pores and elute later; low molecular weight groups can enter all the pores and elute last.

Column Activation

To activate Excsep™ SEC gel columns, rinse with 100% water at a flow rate of 1.0 mL/min for 1–2 hours, followed by equilibration with the mobile phase to prevent "salting out" and other irreversible damage to the column.

Use of Columns

Size exclusion chromatography (SEC) separates molecules based on their size in solution, where retention time depends on how solute molecules permeate the stationary phase's pore structure. However, while this is the ideal separation effect, actual separations are influenced by secondary forces that can affect target separation. Consider the following factors when developing an SEC method:

1.Molecular Weight of Target
Choose the appropriate pore size for the column based on the molecular weight of the analyte.

2.Ionic Strength of Mobile Phase
To minimize secondary retention forces between the packing material and the analyte, adjust the mobile phase's ionic strength. For SEC method development, ionic strength can be adjusted (typically within 0–100 mM) to reduce these forces, improving peak shape and separation.

3. pH of Mobile Phase
   Adjusting the pH of the mobile phase can reduce secondary interactions within the column that may affect the analyte. Excsep™ SEC columns support a pH range of 2–8.

4.Column Length
SEC separation can be enhanced by increasing column length. If a single column does not achieve the desired separation, 

consider connecting two columns in series or using columns with different pore sizes (usually with the larger pore size in front and the smaller in the back).

5.Flow Rate
Lower flow rates generally improve separation and test results. Be mindful of column pressure when adjusting flow rates. Excsep™ SEC columns have a maximum pressure tolerance of 5000 psi (3 µm) and 2500 psi (5–10 µm).

6.Other Variables (Sample Volume, Injection Volume, Column Temperature)
Sample load, injection volume, and column temperature can impact separation. These parameters should be optimized as part of the separation process.

Storage

ExcsepTM SEC columns are recommended to be stored in 90% organic solvents.

Solution for Column Efficiency Reduction

After extended use, samples may accumulate on the inlet frit or within the packing material, potentially leading to increased pressure and peak broadening. The following rinsing procedures may help:

1.High Concentration Neutral Salt Solutions: Use a solution with low pH, such as 0.5 M sodium sulfate.

2.Buffer Solutions with Organic Solvents: Use a buffer (e.g., 50 mM phosphate buffer, pH 7.0) containing 10–20% organic solvent, such as methanol, acetonitrile, or ethanol.

3.Co-Solvents for Strongly Adsorbed Substances: If neutral salt solutions or organic solvents do not provide sufficient results, use co-solvents (e.g., 6–8 M urea or 0.2–0.3% sodium dialkyl sulfate) to remove strongly adsorbed substances (e.g., bound by hydrogen bonding).