Maintenance method for Philomon and ACE columns

Maintenance method for Philomon and ACE columns

Columns are a consumable and therefore have a limited life. For most applications, the column should be continuously injected 500-2000 times (injection), but this will vary depending on the cleanliness of the sample, the pH of the mobile phase, and the use of the guard column ferrule (whether or not a guard column is used).
The practices listed here help maximize the lifetime of silicon-based (silica-based) columns.


1. Column balance : When changing from one mobile phase to another, or in the recovery gradient (gradient cycle), 10-20 column volumes should be required.
The table below shows the column volumes for various column sizes (specifications).
If the change in solvent is less severe (eg, 80% to 20% ACN/water versus ACN to THF (as opposed to ACN to THF)), less volume is required.
The easiest way to check balance is to perform two sample injections.
If left the same, the column is well balanced; if the change is retained, the equilibrium volume should be increased and retested. The balance is related to the volume of the solvent, regardless of time.
Therefore, higher flow rates (flow rates) can reduce the equilibration time.


Approximate column capacity (mL)
Column diameter Column length
50 mm 150 mm 250 mm
2.1 mm 0.1 0.3 0.5
3.2 mm 0.3 0.7 1.2
4.6 mm 0.5 1.5 2.5
10.0 mm 2.4 7.1 11.8
21.2 mm 11.6 34.7 57.8










Second, column rinsing is a simple process that extends the life of the column by cleaning the stubborn (retained) material in the column.
Whenever the column is used daily, remove all buffers (the buffer salts in the column should be removed) and rinse the column with a 100% strong solvent (usually ACN or MeOH, using reversed phase).
The detailed rinsing procedures listed on the next page can effectively restore column performance, but keep in mind that the column is a consumable, so don't expect it to last forever!
Avoid rinsing the reversed-phase column with 100% water (except for the polar group or “AQ” column), because the phase (stationary phase) dewetting will affect the cleaning effect, and the rebalance of the mobile phase column will be very slow. .
For your column, follow the general procedure described below for column rinsing with a specific solvent.
Check the manufacturer's recommendations (manufacturer's instructions) before rinsing to avoid damaging the column.
1. Remove and flip the column (removing the chromatogram and reversing)
2. Connect the column to the pump instead of the detector (no detector)
3. Flush with 10-20 column volumes of solvent at a flow rate no higher than the flow rate of the QC chromatogram
4. If you change the following steps, be sure to use a miscible solvent (solvent miscible) in each successive step.


Reversed phase column (C18, C8, C4, phenyl, CN, 'AQ' type)
a. mobile phase without buffer
b. MeOH or ACN
If metal ions are considered to be the cause of contamination, rinse with 0.05 M EDTA in water, rinse with water, and rinse as described above. Columns using ion-pairing reagents should be used exclusively for ion pair applications.

Unbonded silica gel column (SIL)
a. IPA
b. MeOH
c. Ethyl acetate

Bonded normal phase column (CN, NH 2, diol)
a. chloroform
b. IPA
c. dichloromethane
d. Hexane

Anion exchange column (SAX, WAX)
Water
b. Methanol
c. chloroform
d. Methanol
e. water

Cation exchange column (SCX, WCX)
a. Water (injected 4x200L DMSO when flushing)
b. THF

Protein size exclusion column <br> for weakly retained proteins
a. 0.1M phosphate buffer, pH 3
For strongly retained proteins
a. 100% water to 100% ACN gradient, run for 60 minutes

Third, the reasonable storage of the column helps to extend the life of the column.
The simplest storage step is to remove all buffer from the column and then wash the column with 10-20 column volumes of strong mobile phase solvent (eg MeOH or ACN for reversed phase) to remove stubbornness in the column (strong Retain) substances.
The column is then washed with a storage mobile phase specified by the manufacturer at a capacity of 10-20 column volumes (this information should be provided with the column and detailed in the QC chromatogram).
Finally, securely cap the cap (tighten the plug) to prevent the mobile phase from evaporating.
Do not use buffers or less than about 25% organic solvents for column storage, except for specific conditions that the column manufacturer specifically recommends (such as some ion exchange columns), as they can cause microbial growth. (Save the column to prevent microbial growth)
• Each new project should use a brand new Type B high purity silica column in combination with the highest quality HPLC grade reagents.
• Rinse the HPLC system regularly, remove salts and buffers, and regularly maintain the system to minimize check valves (check valves) and pump seals (seal).

The more thorough the sample cleanup process, the cleaner the sample; at the same time, the probability of occurrence of the sample-related problem is smaller.

• After completing a series of (multiple systems) runs (or in some cases, after each run), a strong solvent wash should be used to help remove stubborn residues (strongly adsorbed) from the column and Minimize column lifetime by minimizing interference in future runs (minimizing interference with future tests).
Columns are not permanently used, but good maintenance reduces unnecessary replacement costs

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