Setting Pressure Limits for Size Exclusion Chromatography

Pressure is generated by the flow through the chromatographic system. For optimal chromatography functionality, it is important to understand the principle of the pressure drop over the different parts of a system (Fig 1.11).

Figure 1.11. Example of the pressure in different parts of a system during run of a column. Note that the pressure values are only used to illustrate the principle.

• ∆p_before does not affect the column.
• The pressure on the column hardware is the sum of ∆p_after and ∆p. Do not exceed the column hardware limit!
• ∆p is individual and needs to be determined for each column.

For more information, refer to the ÄKTATM Laboratory-scale Chromatography Systems Instrument Management Handbook, 29-0108-31.

To protect the column hardware and the packed bed of the chromatographic medium, it is important to set limits that must not be exceeded during the run. There are two important pressure limits that must be taken into consideration:

1. To protect the column hardware: Column hardware pressure limit (Fig 1.12), which is the maximum pressure the hardware can withstand without being damaged. This value is fixed for each column type. Leakage from the column could be a sign of excessive pressure on the column hardware. The column hardware pressure limit is included in the instructions and in UNICORN™ column list for each column type, respectively.
   

Figure 1.12. Column hardware pressure limit is the maximum pressure the column can withstand without damage.

2. To protect the packed bed: Delta pressure (Δp) or maximum pressure over the packed bed is the maximum pressure the packed bed of chromatography medium can withstand without risking gap formation (Fig 1.13) or bed collapse. This value varies depending on conditions. A typical value for Δp or maximum pressure drop over the packed bed is provided for each column type in the instructions and UNICORN column list. Note however that Δp is individual for each column and needs to be determined. The procedure for doing this is described in Instructions 29-0272-71. The packed bed is best protected by controlling the flow rate. Use lower flow rates for high-viscosity solutions and/or low temperature (Table 1.3).

Table 1.3. Example of flow rate limits at different viscosity and temperature, Superose 6 Increase 10/300 GL

Temperature	Flow rate (ml/min)
20 ºC to 25 ºC	Maximum flow rate, water Maximum flow rate, 20% ethanol	1.5 0.75
4 ºC to 8 ºC	Maximum flow rate, water Maximum flow rate, 20% ethanol	0.75 0.35

Figure 1.13. The maximum pressure over the packed bed is the maximum pressure the packed bed of chromatography medium can withstand without gap formation. This is not a fixed value.