Columns should be seen as consumable objects, with a finite lifespan. Columns should last 500-2000 injections for most applications, however, this may vary depending on the cleanliness of the samples, the pH of the mobile phase, and the usage of guard cartridges. The techniques outlined here should aid in extending the usable life of silica-based columns.
Equilibration
When moving from one mobile phase to another or recycling a gradient, the equilibration of the column should take 10-20 column volumes. The column volume is illustrated below for various column dimensions. The less abrupt the shift in solvent (for example, from 80% to 20% ACN/water vs. ACN to THF), the less volume required.
To check for equilibration, do two injections of sample; if the retention is the same, the column was suitably equilibrated; if the retention varies, increase the equilibration volume and try again. Because equilibration is proportional to the volume of solvent, not the time, faster flow rates can shorten equilibration durations.
Estimated Column Volumes (mL)
Column i.d. | 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 |
ALSO READ: Column Restoration Procedures
Column Flushing
Column flushing is a simple process that can increase column lifespan by removing tightly held debris. Remove any buffer and flush the column with 100% of the strong solvent at the conclusion of each day's use (generally ACN or MeOH for reversed-phase methods).
Column is a consumable component and should not be expected to last indefinitely! Except for embedded-polar-group or "AQ" columns, avoid flushing reversed-phase columns with 100% water since phase dewetting will hinder effective cleaning, and column re-equilibration with mobile phase may be sluggish.
Follow the basic process for flushing your column with the specific solvents listed below for your kind of column. It is usually a good idea to verify the manufacturer's guidelines before flushing to avoid damaging the column.
- Disconnect and flip the column
- Connect the column but not the detector to the pump.
- Flush with 10-20 column volumes of solvent at no higher than the flow rate used for the QC chromatogram.
- If you modify the processes below, make sure to use miscible solvents for each stage.
Reversed-Phase Columns (C18, C8, C4, Phenyl, CN, ‘AQ’ type)
a. Mobile phase without buffer
b. MeOH or ACN
- If metal ions are thought to be causing contamination, flush with aqueous 0.05M EDTA, then water, followed by the above sequence. Columns that use ion-pairing reagents should be dedicated to ion-pairing applications.
Reversed-Phase Protein/Peptide Columns
a. Mobile phase without buffer
b. Gradient of 10-90% B; A = 0.1% TFA/water; B = 0.1% TFA/ACN
Unbonded Silica Columns (SIL)
a. IPA
b. MeOH
c. Ethyl acetate
Bonded Normal-Phase Columns (CN, NH2, Diol)
a. Chloroform
b. IPA
c. Methylene chloride
d. Hexane
Anion-Exchange Columns (SAX, WAX)
a. Water
b. Methanol
c. Chloroform
d. Methanol
e. Water
Cation-Exchange Columns (SCX, WCX)
a. Water (inject 4x 200 µL DMSO during flush)
b. THF
Size-Exclusion Columns for Proteins
For weakly retained proteins
a. 0.1M phosphate buffer, pH 3
For strongly retained proteins
a. Gradient of 100% water to 100% ACN in 60 min
Column Storage
Column storage procedures will help to extend the column's life. The most basic storage approach is to remove any buffer from the column and then wash it with 10-20 column volumes of strong mobile phase solvent (e.g., MeOH or ACN for reversed-phase) to remove any firmly held material from the column.
The column should then be flushed with 10-20 column volumes of the storage mobile phase prescribed by the manufacturer (this information should be detailed on the QC test chromatogram originally supplied with the column). Finally, tightly seal the column to avoid mobile phase evaporation.
To avoid microbial development, do not store the columns with buffer or less than roughly 25% organic solvent, unless the column manufacturer specifies otherwise (e.g., certain ion-exchange columns).
Maintenance
The column should preferably be stored in hexane/2-propanol (90/10, v/v) when stored for more than one week. Please pay special attention to the miscibility of the eluents. Use 2-propanol in between eluents of different polarities.
If washing/regeneration steps are required, please use the first 2-propanol, then ethanol (or methanol) at 0.5 ml/min for 3 hours.
To complete the washing process all transitions from one solvent to another should be made via 2-propanol.
Summary
Several typical sources of peak shape difficulties have been investigated, including retention time fluctuation, ghost peaks, and column backpressure. Some of these issues are caused by the sample, while others are caused by the mobile phase and still others by the column or other instrument components. A few healthy behaviors can assist to reduce the incidence of such issues.
- For each new project, use a new Type-B, high-purity silica-based column and the best quality HPLC-grade chemicals.
- Flush the HPLC system on a regular basis to remove salts and buffers, and maintain it on a regular basis to reduce check-valve and pump-seal issues. The more complete the sample cleaning process, the cleaner the sample and the lower the chance of sample-related issues.
- A strong-solvent flush at the conclusion of a series of runs (or after each run in some situations) will assist to eliminate highly held items from the column, minimize interferences in subsequent runs, and increase column lives.
Columns are not indestructible, but with appropriate maintenance, you should get an excellent return on your investment.
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