Section 8 HPLC Column Protection

Problem:

The accumulation of strongly retained material on an HPLC column can dramatically reduce its lifetime. By modifying the packing surface, these retained materials can cause shifts in peak retention, loss of resolution, and efficiency, as well as degradation of peak shape (see Figure 8-1).

Solution:

The best way to protect an analytical column from this type of column fouling is to install a guard column between the injection valve and the analytical column. Guard columns, packed with fully porous particles, having the identical or a slightly less retentive bonded-phase as the analytical column, are recommended. 'These high surface area packings of similar bonded-phase chemistry maximize the protection of the analytical column.

Evaluating the Performance of a Guard Column

Optimally, guard columns should be easy to use and should not degrade the efficiency of the analytical column. To assess whether your guard column preserves the efficiency of the analytical column, run a standard assay with and without the guard column in place. Compare the number of theoretical plates¹ for the same peak in each chromatogram. (This comparison test is more rigorous if the peak has a k value between 1 and 2.) The efficiency of the peak with the guard column should be within 5% of the efficiency of the peak without the guard in place. If a larger decrease in efficiency is observed, call the manufacturer of the guard column for their recommendations.

Minimal Dead Volume Maximizes Guard Column Efficiency

Good, economical guard column performance can be obtained with a cartridge type system, as shown in Figure 8-2. The hardware is reused, and only the guard cartridge is replaced. The new, more efficient design minimizes dead volume so that one holder can be used efficiently with analytical (4.6, 3.0 mm id) and narrow-bore (2.1 mm id) columns. The simple design assembles quickly and easily, making it more likely that a guard column will be used and analytical column lifetime extended.

Replace Guard Columns When ...

A guard column is most effective if it is replaced before the chromatography of the analytical column deteriorates. This can be hard to determine, so many chromatographers select a specific time interval (every week) or sample interval (every 100 samples) for changing the guard column. This is probably the most effective way to minimize contamination of the analytical column. Alternatively, peak shape deterioration and backpressure increases (no more than 10% - see Section 2) can serve as reminders.

Guard Columns or Silica Saturator Columns

Guard columns are used primarily to protect the analytical column from strongly adsorbed sample and matrix. But HPLC column silica is soluble in aqueous mobile phases at higher pH (6 - 9) and temperature, and with high concentrations of buffer salts. Over time the packing dissolves, resulting in increased backpressure, column voids and broad peaks. Guard columns offer some protection from this, because the silica packing will dissolve from front to back; and with frequent guard column changes analytical column life may be preserved. Under very harsh mobile phase conditions (above pH 7 and 40°C, and with a buffer salt concentration > 50 mM), a silica saturator column should also be used. Typically, this is a short column packed with silica that is placed between the pump and the injector. As the mobile phase passes through this column, it dissolves enough silica to saturate (or nearly so) the mobile phase. Then when the mobile phase reaches the analytical column, no additional packing can dissolve. Therefore, column life is extended at high pH. In addition, some HPLC columns are designed for operation at higher pH. Please check the manufacturer's specifications and recommendations when operating a column at higher pH.

Silica saturator columns tan be a disadvantage when doing gradient separations. They can add to the dwell volume and will delay gradient effects on the column.This additional volume must also be taken into account when the method is transferred to assure reproducibility.

These precautions can greatly extend the lifetime of your analytical HPLC columns. If a column becomes contaminated even with these precautions, refer to Section 7 for methods to regenerate your HPLC column.

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