Chromolith® WP 300 Epoxy
Chromolith® WP 300 Epoxy columns are specially designed for the user-specific immobilization of ligands and their later application in HPLC. The unique bimodal pore structure of silica monoliths allows efficient coupling independent of molecule size. The wider mesopores also enable the use of proteins and antibodies as both ligand immobilized on the column, and later analyte separated by an immobilized column.
Potential applications: attach Trypsin to obtain HPLC column-protein digestion reactor, attach a protein for protein-protein interaction analysis, attach any chiral selector to obtain a chiral column, or attach any affinity ligand for a custom made affinity column etc.
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Preparing the Column
The Chromolith® WP 300 Epoxy column is shipped in 100% 2-Propanol. The column has to be washed with 20 CV deionized water before immobilization.
Immobilization via Epoxide Functions
The reaction mechanism for a direct immobilization using the epoxide group is shown in Figure 1. The epoxy ring system enables a nucleophilic attack through a ring opening process leading to a covalent bond between the nucleophile and the primary carbon atom. At the adjacent carbon atom, a hydroxyl group is formed. Epoxides can react with carboxyl, thiol, amine and hydroxyl groups depending on the pH of the medium. It is very common for the reaction between epoxides and amines to form a secondary amine bond between the support and ligand. The use of lyotropic salts in the reaction media enhances the coupling yield. The use of lyotropic salts drives the soluble ligand toward the surface of the support by a salting out effect, enhancing the covalent reaction of epoxide and amine groups at moderate pH.
Figure 1.Reaction between epoxide functions on monolithic surface
Additionally, other parameters influence the coupling yield of epoxide reaction such as the ligand size and concentration, reaction time and temperature. Generally, a higher reaction time will lead to a higher surface coverage and coupling yield. Smaller molecules need higher concentrations than larger molecules (e.g. proteins) to achieve the same degree of surface coverage.
After coupling of the ligand, residual epoxide groups on monolithic surface has to be quenched to avoid undesired backbone interactions with the analytes. Suitable reagents for quenching are 1 M glycine or 1 M urea. If the used ligand is stable at low pH, it is possible to use 150 mM phosphoric acid to hydrolyze remaining epoxide groups. All quenching reactions are finished after at least 30 minutes.
See below for an example immobilization protocol using the epoxide ring system for direct immobilization.
- Connect the column to an HPLC pump (stand-alone system is recommended) and equilibrate the column with 30 CV of 50 mM sodium phosphate + 1.9 M ammonium sulfate pH 8.0 using a flow rate of 2.0 mL/min. The column end is connected directly to the waste. The equilibration step is performed at room temperature.
- Dissolve the desired amount (1–10 mg/mL) of ligand in 25 mL 50 mM sodium phosphate + 1.9 M ammonium sulfate pH 8.0 buffer and recheck the pH value of the ligand solution. Subsequently, connect the solution to the HPLC pump.
- Connect the column end also to the ligand solution and immobilize the ligand to the column and circulate for a maximum of 24 hours at a flow rate of 0.2 mL/min at room temperature.
- Quench the remaining epoxide functions with 1 M glycine for 2 hours at a flow rate of 1.0 mL/min and at room temperature. The column end is directly connected to the waste.
- Finally, equilibrate the column with your working solvent.
Immobilization via Schiff base mechanism
The immobilization via a Schiff base mechanism requires reaction of the epoxide group to form an aldehyde. The aldehydes react with amines forming a Schiff base linkage, which is enhanced under alkaline conditions. The Schiff base linkage is susceptible to hydrolysis and can reform the carbonyl and amine groups. The linkage of both can be stabilized by reduction to a secondary amine bond. As mild reductant, sodium cyanoborohydride can drive the immobilization to completion at neutral pH. Furthermore, it can be used in acidic conditions to quench the residual carbonyl functions at the monolithic surface.
The Schiff base mechanism is much more reactive compared to the described epoxy reaction. Nevertheless, the reaction is affected by several parameters including reaction time, temperature, ligand size and concentration.
Figure 2.Scheme of immobilization via Schiff base mechanism.
See below for an example immobilization protocol using Schiff base mechanism for immobilization of amines.
- Connect the column to an HPLC pump (stand-alone system is best) and hydrolyze the epoxide functional groups to diols with 70 CV of 2% sulfuric acid using a flow rate of 2.0 mL/min at room temperature. The column end is connected directly to the waste.
- Wash the column with at least 15 CV deionized water at a flow rate of 2.0 mL/min. The column end is still connected to the waste.
- The arisen diol groups are oxidized by 100 CV 100 mM sodium periodate in water/methanol 4:1 (v/v) to carbonyl functions at 2.0 mL/min and room temperature. The column end is still connected to the waste.
- Again, wash the column with at least 15 CV deionized water at a flow rate of 2.0 mL/min. The column end is still connected to the waste.
- Dissolve the desired amount (1–10 mg/mL) of ligand and 5 mM sodium cyanoborohydride in 25 mL 50 mM sodium phosphate + 1.9 M ammonium sulfate pH 8.0 buffer and connect the solution to the HPLC pump.
- Connect the column end also to the ligand solution to immobilize the ligand to the column by circulating for a maximum of 24 hours at a flow rate of 0.2 mL/min and room temperature.
- Reduce the remaining carbonyl functions with 20 mM sodium cyanoborohydride, dissolved in 50 mM sodium phosphate pH 3.0, for 60 CV at 2.0 mL/min and room temperature. The column end is directly connected to the waste.
- Finally, equilibrate the column with your working solvent.
Generally, it is possible to perform all activation or immobilization steps at lower temperatures. However, please be aware that lower temperatures prolong the immobilization time.
Use as HPLC column
After immobilization, the column is ready to use for the desired purpose of the immobilized ligand. The type of required solvent or buffers depends on the type of ligand used.
Chromolith® WP Epoxy columns can be used with all commonly used HPLC grade organic solvents, with the following restrictions. The mobile phase should NOT contain more than 50% Tetrahydrofurane (THF), 5% Chlorinated solvent (e.g. Dichloromethane) or 5% Dimethylsulfoxide (DMSO). However pure DMSO can be used as solvent for samples. Buffers, organic modifiers and ion pair reagents present no problems as long as the appropriate pH range is not exceeded. Nevertheless, be careful not to expose the column to conditions which could cause denaturation of your ligand.
Do not exceed the pH range from 1.5 to 7.5 with Chromolith® Widepore columns during analysis. Higher pH will dissolve the silica, creating voids in the column. Lower pH can eventually strip away some of the bonded phase. These defects will cause changes in retention times and loss of resolution.
Column lifetime is highly dependent on the sample and conditions, and cannot be generalized.
For samples with large quantities of contaminants, we recommend to apply one or more sample preparation methods prior to separation (e.g. solid phase extraction, filtration, centrifugation, etc.). Make sure that your samples and the mobile phases are clean and particulate free by using HPLC grade solvents and reagents.
If buffers or other salts are used, a final filtration of the mobile phase should be done with a membrane filter.
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Related Product Resources
Brochure: Chromolith® Monolithic Silica HPLC Columns
Flyer: New Chromolith® WP 300 Epoxy 2 mm HPLC Columns