Separating molecules by molecular size
Size exclusion chromatography (SEC) is a versatile method for separating molecules based on their size. It efficiently purifies and separates high-molecular-weight compounds like viruses, lipids, proteins, and cellular components. In SEC, a sample solution passes through a column packed with porous beads. Larger molecules elute first, as they do not penetrate the pores, while smaller molecules diffuse into the pores, resulting in a delayed elution time. This differential migration allows for precise separation based on molecular size.
The choice of column and packing material is crucial for optimal SEC performance. High-performance SEC columns use small, rigid porous particles made of organic polymers or inorganic silica. Key factors affecting separation quality include surface chemistry, pore size and volume, particle size, column dimensions, sample volume, and flow rate. Selecting the right column and optimizing these parameters ensures effective separation of target molecules.
In Size Exclusion Chromatography (SEC) the column is packed with porous beads and, when the sample is passed through, smaller molecules diffuse through the pores and are thus slowed down while larger molecules avoid the pores and elute faster, allowing for separation of molecules by size.
PolyHYDROXYETHYL A™
PolyHYDROXYETHYL A™ can be used for SEC from the largest proteins to amino acids. This material offers a wide fractionation range with a mildly denaturing mobile phase such as 50mM formic acid.
In SEC, the chaotrope in the mobile phase disrupts the hydrogen bonding between the polymer chains in the coating, increasing their steric radius, making the space between them permeable and permitting the fractionation of solutes as small as dipeptides and amino acids.
Our columns accurately assess the size of:
- The smallest peptides in commercial protein hydrolyzates
- Products from proteasome digestion
- Proteins
The PolyHYDROXYETHYL A™ column is available in four main pore sizes 60Å, 200Å, 300Å and 1000Å.
| Pore Diameter | Denaturing | Nondenaturing |
|---|---|---|
| 60Å | 20-600 | 60-10,000 |
| 200Å | 20-1,600 | 200-40,000 |
| 300Å | 20-40,000 | 300-100,000 |
| 500Å | 20-100,000 | 600-500,000 |
| 1000Å | 20-500,000 | 1,000-1,000,000 |
The HEA columns may also be used with volatile mobile phases.
FEATURED APPLICATION NOTE:
High-Throughput Screening of Small Molecule Combinatorial Libraries
Screen combinatorial libraries of up to 2,000 components per run to identify small molecules that bind with high affinity to a large target entity, without the need for derivatization or specialized equipment.
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