Size Exclusion Chromatography (SEC) separates compounds based on the size of the molecules in solution, where larger molecules are excluded by finer pores in the packing of the column. SEC involves gel permeation chromatography (GPC) when an organic solvent is used as the mobile phase and a synthetic polymer packing is used. SEC is referred to as gel filtration chromatography when an aqueous buffer is used as the mobile phase and a hydrophilic column packing is used to separate biopolymers such as proteins. Gel filtration chromatography can be used as a pretreatment tool to separate biologically active substances (often equivalent to other chromatographic techniques in multiple purification steps), or as a tool to obtain molecular information on solutes, including molecular size or shape, aggregation State, kinetic parameters of biopolymer binding to its ligands.
In the past, gel filtration chromatography generally used soft gels such as dextran, agarose or polyacrylamide as fillers. For compressible packing, a suitable gravity or low pressure pump must be selected according to the flow rate of the mobile phase. Soft gels can be stabilized by cross-linking, so that they can withstand higher flow rates and pressures of several MPa. Rigid supports commonly used in gel filtration chromatography are: silica matrix modified with hydrophilic stationary phase or cross-linked organic polymers. These substances can be used in HPLC systems, and their mechanical strength can withstand pressures of tens of MPa or more.
Size Exclusion Chromatography (SEC), as the name implies, its basic principle is that when the target analyte enters the gel permeation chromatography column, large-volume molecules cannot enter the pores because they are larger than the pores of the gel particles, and can only enter the pores between the gel particles. It flows through the gaps between the particles and is first eluted out; the molecular part of the medium volume can enter the pores of the gel particles and flow through the gaps between the particles, and the order of elution is between the large-volume molecules and the small molecules; Molecules with smaller volume are smaller than the pores of the gel particles, and can enter the pores of the gel particles, and continue to enter and diffuse in the pores of the gel particles and the gaps between the particles, with the slowest speed, so they are finally washed out. of. When the instrument model and the conditions required for the experiment are determined, the eluted volume of the compound is related to its molecular weight. The larger the molecular weight, the smaller the eluted volume. The specific separation principle is shown in the figure.
Xtimate® SEC chromatographic column is a silica-based size exclusion series column launched by Welch. Its chromatographic packing is a hydrophilic polymer bonded to the surface of silica microspheres with high purity and good stability. Welch Materials uses special surface modification technology to ensure good stability and batch reproducibility of the filler.
Size exclusion chromatography separates according to the molecular weight in solution, and its retention time depends on the relative permeability of the solute molecules in and out of the pore structure of the stationary phase. However, this is a separation situation under ideal conditions, and there are other secondary forces in the chromatographic column that affect the separation of the target.
Therefore, the following factors need to be considered when developing methods with Xtimate® SEC columns:
1. Molecular mass
The chromatographic column with suitable pore size can be selected according to the molecular weight of the target.
2. Sample and mobile phase
To avoid column clogging, all samples and solvents, including buffer salts, must be filtered with 0.45 μm or 0.22 μm filters prior to use. Xtimate® SEC can be used with water or a mixture of organic solvents and water, and is also compatible with most buffered saline solutions. The mobile phase should be degassed before use, otherwise large fluctuations in column pressure and baseline may occur. At this time, a larger flow rate can be used to flush the chromatographic column for 2-5min. For example, for a 7.8mm*300mm chromatographic column, a flow rate of 1.25mL/min can be used.
3. Ionic strength
There are inevitably other secondary forces in the chromatographic column. In order to minimize the secondary forces between the packing material and the analyte, the ionic strength of the mobile phase must be adjusted. NaCl is a commonly used salt in SEC separation, which can improve peak shape and separation effect by adjusting ionic strength and reducing secondary forces.
The pH adjustment of the mobile phase is more to reduce the influence of the secondary action in the chromatographic column on the analyte, so it is necessary to select an appropriate pH during the measurement process. For best separation and longevity, mobile phases with a pH in the range of 2-7.5 are recommended.
Although Xtimate® SEC can be used at pressures up to 3000psi, normal operating pressures should be below 1500psi. Prolonged operation at high pressure can damage the column and infusion pump. Since the pressure is derived from the flow rate, the maximum flow rate will be limited by the pressure the system can handle. In general, the column pressure will gradually increase with the age of the column.
6. Flow velocity
Try to use a low flow rate test as much as possible, which can improve the resolution and obtain better test results. For chromatographic columns with inner diameters of 4.6mm and 7.8mm, it is generally recommended that their normal operating flow rates be 0.1-0.4mL/min and 0.1-1.25mL/min, respectively.
7. Length of column
The SEC resolution can be improved by increasing the length of the chromatographic column. Therefore, in the separation process, when one chromatographic column cannot achieve the separation effect, two chromatographic columns can be considered in series, or even chromatographic columns with different pore diameters can be connected in series (Note: generally larger The aperture is in the front, the small aperture is in the back). Such conditions also typically result in delayed peak times and increased system pressure.
8. Column temperature
The maximum operating temperature is 80°C. In order to obtain the longest service time, the optimal operating temperature is 10-30℃. Prolonged operation at high temperature (>80°C) can also damage the column, especially at high pH (>7.5).
9. Daily maintenance
As the number of uses increases, some samples may adsorb to the inlet frit or packing. When the accumulation reaches a certain level, the pressure will increase, accompanied by abnormal peak shape and other phenomena. Therefore, it is necessary to frequently pay attention to the flushing and maintenance of the chromatographic column during daily use to achieve the best separation effect and prolong the service life.