Occasionally, samples contain components that will irreversibly 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:
- Asymmetry Factor
- Retention Time
- Resolution
- Theoretical Plates
You do not necessarily have to run a standard test probe to monitor your column. If you have a well-resolved peak, this can be used to check column performance as well. 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.
- Clean your column in the reverse flow direction. [Since the 'contamination' takes place first at the top of the column, it will have to travel a shorter distance to exit the column when you turn it upside down.]
- Do not connect the column to the detector. [No need to have the 'dirt' get in contact with the detector cell.]
- Run the column at half of the maximum recommended flow rate, taking special care to monitor the pressure as the cleaning solution may be of different viscosity than your normal mobile phase.
- 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.
- If multiple cleaning solutions must be used, always rinse the column between cleaning solutions with 3-5 CV of Milli-Q or DI water.
- 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
TSKgel® SW and SWXL
- Turn the column in reverse flow direction and run at half the maximum flow rate.
- Clean with 5 column volumes (CV) of 1 mol/L sodium chloride (pH 7.0)
- Clean with 5 CV of Milli-Q or DI water.
- Clean with 5 CV of 20% acetonitrile.
- Clean with 5 CV of Milli-Q or DI water.
- Turn column in normal flow direction and equilibrate in mobile phase for at least 45 minutes.
TSKgel® PW and PWXL
- Turn column in reverse flow direction and run at half the maximum flow rate.
- Clean with 5 CV of 1 mol/L sodium chloride (pH 7.0)
- Clean with 5 CV of Milli-Q or DI water.
- Clean with 5 CV of 20% acetonitrile.
- Clean with 5 CV of Milli-Q or DI water.
- Turn column in normal flow direction and equilibrate in mobile phase for at least 45 minutes.
Ion Exchange and TSKgel® SW-Type
- High concentration salt (e.g. 0.5 mol/L - 1.0 mol/L) of your normal run buffer
- Buffered solutions at low pH (e.g. 2 - 3)
- Water soluble organic (MeOH, ACN, EtOH,10% - 20%) in aqueous buffer
- Urea (8 mol/L) or non-ionic surfactant in buffer solution.
Ion Exchange and TSKgel® PW-Type
- 0.1 mol/L - 0.2 mol/L NaOH
- 20% - 40% aqueous acetic acid
- Water soluble organic (MeOH, ACN, EtOH,10% - 20%) in aqueous buffer
- Urea (8 mol/L) or non-ionic surfactant in buffer solution.
Ion Exchange and TSKgel® STAT-Type
- 0.1 mol/L NaOH
Hydrophobic Interaction
- 0.1 mol/L - 0.2 mol/L NaOH
- 20% - 40% aqueous acetic acid
Reversed Phase and TSKgel® SW-Type
- Acetonitrile or methanol
- Gradient from 10% - 100% acetonitrile or methanol in 0.05% trifluoroacetic acid
Reversed Phase and TSKgel® PW-Type
- Acetonitrile or methanol
- 0.1 mol/L - 0.2 mol/L NaOH
- 20% - 40% aqueous acetic acid
Sometimes a column dries out unintentionally, either because the mobile phase ran out and air was pumped through the column or because the endfittings were not air-tight during long-term storage. This 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:
- Connect the column to your LC system in reverse flow direction.
- Make sure not to connect the column to the detector during this procedure.
- Pump a filtered mobile phase of 20% methanol in distilled, deionized water through the column at half of the recommended maximum flow rate. Use 60% methanol in case of reversed phase columns.
- Continue this procedure until you are confident that the column has been rehydrated. Rehydration can take several hours, depending on the column size.
- Connect the column to your LC system in normal flow direction.
- Equilibrate with your normal mobile phase.
- Perform the recommended QC tests to ensure that the column is performing properly.
TOYOPEARL® and TSKgel® Resins
With the exception of some affinity resins, TOYOPEARL® and TSKgel® resins were designed to withstand sanitization conditions commonly used in large scale manufacturing.
General Resin Cleaning Regimen
A general use cleaning procedure is as follows: 3- 5CV of 0.1 - 0.5 mol/L sodium hydroxide, followed by 3 - 5 CV of 0.5-1 mol/L sodium chloride before column equilibration. TOYOPEARL® and TSKgel® resins can withstand caustic for well up to 8 hrs at a time. Be sure to operate at reasonable flow rates to avoid over pressuring as the resin will swell in the presence of base. Maximum pressure limit for TOYOPEARL® resins is 3 bar (45 psi) and for TSKgel® resins is 20 bar (300 psi).
Removing Protein
In some cases, protein may be tightly bound to the resin.
- In cases where protein is bound ionically, follow the hydroxide wash with 5 CV of water then 3 - 5 CV of 0.1 -0.5 mol/L hydrochloric acid before washing with salt.
- In cases where protein is bound hydrophobically to either HIC or SEC resin, wash the resin with 3 - 5CV of water, followed by 3 - 5 CV of 20% methanol or acetonitrile. Rinse with 3-5 CV of water before following the general use cleaning procedure.
Cleaning Affinity Resins
In regards to the affinity resins, the AF-Chelate resin may be cleaned following the general use cleaning procedure while the more ligand sensitive resins should be cleaned with any combination of salt, chaotrope (Urea/ Guanidine HCl), or detergents (Triton/ Tween).
Ca⁺⁺Pure-HA® Resin
Ca⁺⁺Pure-HA® becomes increasingly stable at elevated pH. Hydroxide, either NaOH or KOH, can be used as a sanitizing agent. As with polymer based chromatography media, 1.0 mol/L NaOH is used widely. Contact duration can be the same as with polymer based media, with one hour contact time being common. Unlike many polymer-based media however, stability of Ca⁺⁺Pure-HA in high concentrations of hydroxide is essentially indefinite.
Ca⁺⁺Pure-HA® exposure to concentrated hydroxide should not immediately follow highly concentrated phosphate. It is recommended to rinse the column before introduction of hydroxide with 1–2 column volumes of buffer containing less than 20 mmol/L phosphate. In most cases, the column equilibration buffer serves this purpose.