The Superdex 200 Increase 10/300 GL column is a laboratory tool specifically designed for high-resolution size exclusion chromatography (SEC) of biomolecules. This technique separates proteins and other macromolecules primarily based on their hydrodynamic radius, effectively separating different sizes of molecules as they pass through a porous matrix. The column is widely used for both analytical purposes, such as checking sample purity, and small-scale preparative purification in the milligram range. Its design provides superior resolution and reduced run times compared to earlier generations of size exclusion columns.
Understanding the Superdex 200 Media
The separation matrix is a composite material made from cross-linked agarose and dextran, offering mechanical stability and low non-specific interaction with biological samples. This structure provides high chemical stability, allowing for an operational pH range of 3 to 12, suitable for most aqueous buffers. The “Increase” designation signifies the use of smaller, more rigid resin beads (median particle size 8.6 µm), which contributes to the column’s enhanced resolution.
The column dimensions are 10 millimeters internal diameter (ID) by 300 millimeters in length, resulting in a total bed volume of 24 mL. The fractionation range for globular proteins is 10 kilodaltons (kDa) to 600 kDa. This range makes it suited for separating monomers, dimers, and aggregates of common proteins, such as monoclonal antibodies (100 kDa to 300 kDa). The exclusion limit, above which all molecules elute together, is approximately 1.3 megadaltons (MDa). The “GL” designation indicates the column is constructed from glass.
Initial System Preparation and Equilibration
Before first use or after storage, the system and column must be prepared for optimal performance. First, ensure the chromatography system is plumbed correctly, connecting the flow path while observing pressure limits. The column is shipped in a storage solution, typically 20% ethanol, which must be completely removed before introducing the running buffer.
The system lines and column should first be flushed with a non-viscous solution, such as room-temperature water, at a low linear velocity to gently displace the storage solution. Subsequently, introduce the running buffer (eluent). All buffers must be filtered through a 0.22 micrometer filter and thoroughly degassed to prevent air bubbles, which severely disrupt the packed bed and cause a loss of resolution.
Equilibration saturates the column matrix with the running buffer until the chemical environment is stable. This requires running at least two column volumes (CV) of eluent through the column at the recommended operating flow rate. Stability is monitored by observing a flat and consistent UV detector baseline. Additionally, the conductivity and pH of the buffer exiting the column must match the buffer entering the system, confirming the column is ready for sample injection.
Optimal Separation Parameters and Sample Handling
Selecting the appropriate operating flow rate directly influences separation resolution and run time. The recommended operating flow rate is 0.75 mL/min at room temperature, although optimal resolution for complex samples is often achieved between 0.25 to 0.75 mL/min. The maximum flow rate must not exceed 1.8 mL/min. Higher viscosity buffers or lower temperatures require a proportional reduction in flow rate to prevent excessive back pressure that could damage the column bed.
SEC is an isocratic technique, meaning the buffer composition remains constant because separation is based purely on size exclusion. The running buffer must maintain the stability and solubility of the target molecule. It typically includes 150 to 300 mM sodium chloride to minimize unintended ionic interactions with the matrix. Mild detergents or chaotropic agents may be necessary for membrane proteins or aggregation-prone proteins, but this requires balancing against potential viscosity changes and flow rate reduction.
Proper sample preparation is necessary to maintain column performance and resolution. The sample volume should be small relative to the column volume to ensure sharp peaks, with a recommended range of 25 to 500 µL (0.1% to 2% of the column volume). Protein concentrations below 10 mg/mL often yield the best resolution, though up to 50 mg/mL may be tolerated. Samples must be clarified immediately before injection, either by centrifugation (10,000 x g for 10 minutes) or filtration (0.22 µm filter), to remove particulates.
Column Maintenance and Long-Term Storage
Routine maintenance ensures the longevity and consistent performance of the column. A cleaning-in-place (CIP) protocol should be performed regularly, typically after every 10 to 20 separation cycles, or immediately if resolution loss or increased back pressure occurs. Cleaning involves washing the column with a solution designed to remove adsorbed contaminants like precipitated proteins or lipids.
A common CIP solution is 0.5 M sodium hydroxide, applied for a short period (e.g., one column volume), followed immediately by a water rinse and re-equilibration with the running buffer. For severe contamination, agents like 30% acetonitrile or 0.1 M hydrochloric acid may be used in short cycles, but the column must never be stored in these aggressive solutions. Reversing the flow direction during cleaning is recommended to help dislodge trapped material from the column inlet.
Column performance should be periodically checked using a standard reference sample, such as acetone, to calculate the number of theoretical plates and peak asymmetry. This performance check provides a quantitative measure of separation efficiency and helps detect early signs of bed degradation or fouling.
For long-term storage, the column must be thoroughly washed with two column volumes of water. It should then be equilibrated with at least two column volumes of a bacteriostatic solution, typically 20% ethanol, before storage at 4–8°C. The column must never be allowed to dry out, as the storage solution prevents microbial growth that could destroy the matrix.