TECHNICAL SUPPORT

Column Rehydration and Cleaning

REHYDRATION

Dehydration of TSKgel liquid chromatography columns can result from improper use or during long-term storage. The plugs supplied with TSKgel glass columns do not always provide an airtight seal, which may allow the column to dry out over time. The same can happen to any column, stainless steel or glass, if the plugs are not tightened or if air inadvertently is pumped into the column during use. It is easier to detect dehydration in glass columns because the dry packing will appear to pull away from the the column walls. This condition can be remedied by using the following procedure:

1. Connect the column to your LC system in reverse flow direction.
2. Do not connect the column to the detector.
3. Pump a filtered mobile phase of 20% methanol in distilled, deionized water over the column at half of the recommended maximum flow rate. Please note that reversed phase columns require 60% methanol.
4. Continue this procedure until you are confident that the column has been rehydrated. Rehydration can take several hours, depending on the column size.
5. Connect the column to your LC system in proper flow direction.
6. Equilibrate with your normal mobile phase.
7. Perform the recommended QC tests to ensure that the column is performing properly.


CLEANING

Occasionally, samples are run which adsorb onto the packing material. When this occurs, it is time to clean your column. Generally, if one of the performance characteristics of your column changes by 10% or more, it is prudent to clean your column. These performance characteristics are:

1. Asymmetry Factor
2. Retention Time
3 .Resolution
4. Theoretical Plates

You do not necessarily have to run a standard test probe to monitor your column. If you have a well-resolved peak, the column can be used. To use a sample component, you must establish baseline data when the column is new and performing well. After establishing that the column is performing properly, using standard test probes, calculate the asymmetry factor, theoretical plates and resolution of one or more of your sample components. Also note the retention time. This becomes your "baseline test mix" with which you can later compare.

When you clean the column, there are a few basic rules to follow. These rules apply regardless of what type of TSKgel column you are running.

1. Clean your column in the reverse flow direction.
2. Do not connect the column to the detector.
3 .Run the column at half of the maximum recommended flow rate, taking special care to monitor the pressure as the cleaning solutions may be of different viscosities than your normal mobile phase.
4 .If you are cleaning with a high or low pH solution, make certain that the rest of your chromatographic system (pump, pump seals, injector, etc.) is compatible.
5. If multiple cleaning solutions must be used, always rinse the column between cleaning solutions with 3-5 cv of Milli-Q or DI water.
6. If the cleaning solutions listed below do not improve the resolution of the column, then urea or non-ionic surfactant may be tried.

Cleaning Solutions

SW and SWXL
1. Turn column in reverse flow direction and run at half the maximum flow rate.
2. Clean with 5 column volumes (cv) of 1 M Sodium Chloride (pH 7.0)
3. Clean with 5 cv of Milli-Q or DI water.
4. Clean with 5 cv of 20% Acetonitrile.
5. Clean with 5 cv of Milli-Q or DI water.
6. Turn column in normal flow direction and equilibrate in mobile phase for at least 45 minutes.


PW and PWXL
1. Turn column in reverse flow direction and run at half the maximum flow rate.
2. Clean with 5 column volumes (cv) of 1 M Sodium Chloride (pH 7.0)
3. Clean with 5 cv of Milli-Q or DI water.
4. Clean with 5 cv of 20% Acetonitrile.
5. Clean with 5 cv of Milli-Q or DI water.
6. Turn column in normal flow direction and equilibrate in mobile phase for at least 45 minutes.


Ion Exchange, SW Type
1. High concentration salt (e.g. 0.5 M - 1.0 M) of your normal run buffer
2. Buffered solutions at low pH (e.g. 2.0 - 3.0)
3. Water soluble organic (MeOH, ACN, EtOH,10% - 20%) in aqueous buffer
4. Urea (8 M) or non-ionic surfactant in buffer solution.

Ion Exchange, PW Type
1. 0.1 M - 0.2 M NaOH
2. 20% - 40% aqueous acetic acid
3. Water soluble organic (MeOH, ACN, EtOH,10% - 20%) in aqueous buffer
4. Urea (8 M) or non-ionic surfactant in buffer solution.

Hydrophobic Interaction
1. 0.1 M - 0.2 M NaOH
2. 20% - 40% aqueous acetic acid

Reversed Phase, SW Type
1. Acetonitrile or methanol
2. Gradient from 10% - 100% acetonitrile in 0.05% trifluoroacetic acid

Reversed Phase, PW Type
1. Acetonitrile or methanol
2. 0.1 M - 0.2 M NaOH
3. 20% - 40% aqueous acetic acid