WorkBeads™ Macro SEC resin is used for preparative size exclusion chromatography (SEC) in laboratory and process scale purification of proteins, virus and other biomolecules by utilizing differences in their size. This resin is based on agarose, a biopolymer suitable for separation of biomolecules.
WorkBeads resins are cross-linked using a proprietary method that results in a very rigid, highly porous and physically stable matrix
Although the general recommendation for SEC is to use low flow rate for best purification, the rigidity and tight particle size distribution allows purification of viruses and other large substances at high flow rate for fast processing and high yields.
Size Exclusion Chromatography (SEC) is a simple and reliable technique for separation of molecular components according to their size. The technique relies on a separation resin of porous beads, packed closely together in a column. The packed column is prepared by equilibration with a suitable running buffer, usually an aqueous buffer, before sample loading.
In SEC, large substances will elute earlier than small molecules, since large substances do not enter the pores of the beads or they only enter a fraction of the pores. Smaller substances can enter a larger fraction of the pores and they will therefore elute later. Intermediate size substances will enter the bead pores to different degrees, thus being eluted at different elution volumes. The eluent (buffer) that is used for equilibration of the column will enter completely into the pores of the beads. This means that the substances to be separated will be eluted in the eluent used for equilibration, thus allowing buffer exchange or salt removal (or addition). In addition to size, other characteristics such as shape, charge, hydrophobicity and interactions between substances or substances and resin, may affect the elution volume, thus the separation. Optimization of chromatography conditions (buffer composition, flow and sample volume) may be needed to obtain the required purification. The column is prepared for the next run by passing at least one column volume (CV) of buffer through the column. This will also ensure removal of possible remaining low molecule weight substances from the sample (e.g. salt, buffer, and low Mr impurities).
SEC is usually applied as the last purification step (polishing step) since it is limited in flow and sample volume, and since it allows removal of aggregates of the target substance, buffer exchange and salt removal.
Table 1. Main characteristics of WorkBeads MACRO SEC resins.
Rigid, highly cross-linked agarose
|10 - 30 000 kD
30 000 kD
Average particle size² (Dv50)
Recommended flow rate³
|15 - 150 cm/h
Max flow rate⁴˒⁵
Compatible with all standard aqueous buffers used for protein purification. Should not be stored at low pH for prolonged time.
|2 – 13
2 to 25 °C in 20% ethanol
¹. Globular proteins.
².The median particle size of the cumulative volume distribution.
³.The flow rate is important for the resolution and a lower flow rate often gives an increased resolution. A higher flow rate can be used during equilibration to speed up the separation.
⁴.Determined in water using a 25 x 200 mm column.
⁵.Note: Make sure that the column hardware max pressure is not exceeded.